Polynucleotide encoding a novel TRP channel family member, TRP-PLIK2, and splice variants thereof

ABSTRACT

The present invention provides novel polynucleotides encoding TRP-PLIK2 polypeptides, fragments and homologues thereof. The present invention also provides polynucleotides encoding variants and splice variants of TRP-PLIK2 polypeptides, TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2d, respectively. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel TRP-PLIK2, TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2d polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

[0001] This application claims benefit to provisional application U.S.Ser. No. 60/292,599 filed May 22, 2001; and to provisional applicationU.S. Ser. No. 60/362,944, filed Mar. 8, 2002. The entire teachings ofthe referenced applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention provides novel polynucleotides encodingTRP-PLIK2 polypeptides, fragments and homologues thereof. The presentinvention also provides polynucleotides encoding variants and splicevariants of TRP-PLIK2 polypeptides, TRP-PLIK2b, TRP-PLIK2c, andTRP-PLIK2d, respectively. Also provided are vectors, host cells,antibodies, and recombinant and synthetic methods for producing saidpolypeptides. The invention further relates to diagnostic andtherapeutic methods for applying these novel TRP-PLIK2, TRP-PLIK2b,TRP-PLIK2c, and TRP-PLIK2d polypeptides to the diagnosis, treatment,and/or prevention of various diseases and/or disorders related to thesepolypeptides. The invention further relates to screening methods foridentifying agonists and antagonists of the polynucleotides andpolypeptides of the present invention.

BACKGROUND OF THE INVENTION

[0003] Intracellular Ca²⁺ plays a pivotal role in various cellfunctions, ranging from exocytosis and contraction to gene expressionand cell differentiation, proliferation and apoptosis. Human mutationsin the genes involved in intracellular Ca²+ handling result in visualdefects, diabetes mellitus, disorders in the skin, skeletal-muscle,nervous, cardiac and vascular systems (reviewed by Missiaen et al.,2000). In addition to the well characterized voltage-dependent Ca²⁺channels, Ca2+pumps and Ca²⁺-permeable ligand-gated channels, TRPC(Transient Receptor Potential Channels) is an emerging class ofCa²+-permeable cation channel superfamily. All of the channels in thisfamily contain a six-trans-membrane domain although various cellularmechanisms have been implicated in their functions (reviewed byHarteneck et al., 2000).

[0004] The first member of this family, DTRP, was identified fromDrosophila mutants trp whose photoreceptors failed to generate asustained receptor potential in response to intense sustained light. Themutant fly showed a reduced Ca²⁺ selectivity of the light response andthe channel activity of DTRP depended on PLC activation was alsodemonstrated. Later, many mammalian homologues have been cloned andbased on their homology, they are divided into three subfamilies: short(s), osm (o) and long (1). The sTRPC subfamily includes TRPI-7. Althoughthe specific physiological function of each isoform remains to beassigned, it is generally believed that they may be involved in Ca²⁺entry after activation of receptors coupling to PLC. The TRP2 isspecifically expressed in vomeronasal organ and involved in pheromonesensory signaling (Liman, et al., 1999). TRPI and TRP6 are functioned invascular smooth muscle cells and may play a role in controlling smoothmuscle tone, arteriosclerosis and neointimal hypoerplasia (Inoue et al.,2001; Xu & Beech, 2001). It has been shown that TRP4-/- mice lack anendothelial store-operated Ca²⁺ current, which leads to reducedagonist-dependent vasorelaxation (Freichel et al., 2001).

[0005] The first member of oTRPC Subfamily is OSM-9 cloned from C.elegans. It is involved in responses to odorants, high osmotic strength,and mechanical stimulation. Recently, several mammalian homologuesincluding vanilloid receptor (VR1) and vanilloid receptor-like receptor(VRL-1), which may have functions in pain and heat perception (Caterina,1999; Caterina et al., 2000). VR1 has also been shown to be the receptorof anandamide and mediating its vasodilation effect (Zygmunt et al.,1999). OTRPC4 is an osmotically activated channel and a candidateosmoreceptor, may be involved in regulation of cellular volume(Strotmann et al., 2000). CaTgi & ECaC1 may be thecalcium-release-activated calcium channel and involved in Ca²+reabsorption in intestine and kidney (Peng, et al, 1999; Yu et al.,2001).

[0006] The function of the lTRPC is less clear. The cloned mammalianITRPC includes melastatin 1/MLSN1/LTRPC1, MTR1/LTRPC5, TRPC7/LTRPC2 andTRP-P8. It is known that melastatin I is down regulated in metastaticmelanomas (Duncan et al., 1998) and MTRI is associated withBeckwith-Wiedemann syndrome and a predisposition to neoplasias (Prawittet al., 2000). TRPC7 is mapped to the chromosome region linked tobipolar affective disorder, nonsyndromic hereditary deafness, Knoblochsyndrome and holosencephaly (Nagamine et al., 1998). TRP-P8 is aprostate-specific gene and up-regulated in prostate cancer and othermalignancies (Tsavaler et al., 2001). A recently clonedTRP-PLIK/hSOC-2/hCRAC-1 exhibits a very interesting feature in that itis a bi-functional protein with kinase and ion channel activities(Runnels et al., 2001). Additionally, a very long TRPC homologue NOMPCwas found in Drosophila and C. elegans. NOMPC was identified as amechanosensitive channel that can detect sound, pressure or movementchanges (Walker et al., 2000).

[0007] Characterization of the TRP-PLIK2 polypeptide of the presentinvention led to the determination that it is involved in the modulationof the NFkB pathway, either directly or indirectly.

[0008] The fate of a cell in multicellular organisms often requireschoosing between life and death. This process of cell suicide, known asprogrammed cell death or apoptosis, occurs during a number of events inan organisms life cycle, such as for example, in development of anembryo, during the course of an immunological response, or in the demiseof cancerous cells after drug treatment, among others. The final outcomeof cell survival versus apoptosis is dependent on the balance of twocounteracting events, the onset and speed of caspase cascade activation(essentially a protease chain reaction), and the delivery ofantiapoptotic factors which block the caspase activity (Aggarwal B. B.Biochem. Pharmacol. 60, 1033-1039, (2000); Thomberry, N. A. andLazebnik, Y. Science 281, 1312-1316, (1998)).

[0009] The production of antiapoptotic proteins is controlled by thetranscriptional factor complex NF-kB. For example, exposure of cells tothe protein tumor necrosis factor (TNF) can signal both cell death andsurvival, an event playing a major role in the regulation ofimmunological and inflammatory responses (Ghosh, S., May, M. J., Kopp,E. B. Annu. Rev. Immunol. 16, 225-260, (1998); Silverman, N. andManiatis, T., Genes & Dev. 15, 2321-2342, (2001); Baud, V. and Karin,M., Trends Cell Biol. 11, 372-377, (2001)). The anti-apoptotic activityof NF-kB is also crucial to oncogenesis and to chemo- andradio-resistance in cancer (Baldwin, A. S., J. Clin. Inves. 107,241-246, (2001)).

[0010] Nuclear Factor-kB (NF-kB), is composed of dimeric complexes ofp50 (NF-kB1) or p52 (NF-kB2) usually associated with members of the Relfamily (p65, c-Rel, Rel B) which have potent transactivation domains.Different combinations of NF-kB/Rel proteins bind distinct kB sites toregulate the transcription of different genes. Early work involvingNF-kB suggested its expression was limited to specific cell types,particularly in stimulating the transcription of genes encoding kappaimmunoglobulins in B lymphocytes. However, it has been discovered thatNF-kB is, in fact, present and inducible in many, if not all, cell typesand that it acts as an intracellular messenger capable of playing abroad role in gene regulation as a mediator of inducible signaltransduction. Specifically, it has been demonstrated that NF-kB plays acentral role in regulation of intercellular signals in many cell types.For example, NF-kB has been shown to positively regulate the humanbeta-interferon (beta-IFN) gene in many, if not all, cell types.Moreover, NF-kB has also been shown to serve the important function ofacting as an intracellular transducer of external influences.

[0011] The transcription factor NF-kB is sequestered in an inactive formin the cytoplasm as a complex with its inhibitor, IkB, the mostprominent member of this class being IkBa. A number of factors are knownto serve the role of stimulators of NF-kB activity, such as, forexample, TNF. After TNF exposure, the inhibitor is phosphorylated andproteolytically removed, releasing NF-kB into the nucleus and allowingits transcriptional activity. Numerous genes are upregulated by thistranscription factor, among them IkBa. The newly synthezised IkBaprotein inhibits NF-kB, effectively shutting down furthertranscriptional activation of its downstream effectors. However, asmentioned above, the IkBa protein may only inhibit NF-kB in the absenceof IkBa stimuli, such as TNF stimulation, for example. Other agents thatare known to stimulate NF-kB release, and thus NF-kB activity, arebacterial lipopolysaccharide, extracellular polypeptides, chemicalagents, such as phorbol esters, which stimulate intracellularphosphokinases, inflammatory cytokines, IL-1, oxidative and fluidmechanical stresses, and Ionizing Radiation (Basu, S., Rosenzweig, K,R., Youmell, M., Price, B, D, Biochem, Biophys, Res, Commun.,247(l):79-83, (1998)). Therefore, as a general rule, the stronger theinsulting stimulus, the stronger the resulting NF-kB activation, and thehigher the level of IkBa transcription. As a consequence, measuring thelevel of IkBa RNA can be used as a marker for antiapoptotic events, andindirectly, for the onset and strength of pro-apoptotic events.

[0012] Using the above examples, it is clear the availability of a novelcloned transient receptor potential channel family provides anopportunity for adjunct or replacement therapy, and are useful for theidentification of transient receptor potential channel agonists, orstimulators (which might stimulate and/or bias transient receptorpotential channel function), as well as, in the identification oftransient receptor potential channel inhibitors. All of which might betherapeutically useful under different circumstances.

[0013] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells, in addition to their usein the production of TRP-PLIK2, TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2dpolypeptides using recombinant techniques. Synthetic methods forproducing the polypeptides and polynucleotides of the present inventionare provided. Also provided are diagnostic methods for detectingdiseases, disorders, and/or conditions related to the TRP-PLIK2,TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2d polypeptides and polynucleotides,and therapeutic methods for treating such diseases, disorders, and/orconditions. The invention further relates to screening methods foridentifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

[0014] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe TRP-PLIK2 protein having the amino acid sequence shown in FIGS. 1A-G(SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone,TRP-PLIK2 (also referred to as LTRPC6, BAC57, AL354795, and/or gene 95)deposited as ATCC Deposit Number PTA-4175 on Mar. 21, 2002.

[0015] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe TRP-PLIK2b protein having the amino acid sequence shown in FIGS.2A-G (SEQ ID NO:4) or the amino acid sequence encoded by the cDNA clone,TRP-PLIK2b (also referred to as LTRPC6, BAC57, AL354795, and/or gene 95splice variant).

[0016] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe TRP-PLIK2c protein having the amino acid sequence shown in FIGS.3A-G (SEQ ID NO:6) or the amino acid sequence encoded by the cDNA clone,TRP-PLIK2c (also referred to as LTRPC6, BAC57, AL354795, and/or gene 95splice variant).

[0017] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe TRP-PLIK2d protein having the amino acid sequence shown in FIGS.4A-G (SEQ ID NO:8) or the amino acid sequence encoded by the cDNA clone,TRP-PLIK2d (also referred to as LTRPC6, BAC57, AL354795, and/or gene 95splice variant).

[0018] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells, in addition to their usein the production of TRP-PLIK2, TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2dpolynucleotides or polypeptides using recombinant techniques. Syntheticmethods for producing the polypeptides and polynucleotides of thepresent invention are provided. Also provided are diagnostic methods fordetecting diseases, disorders, and/or conditions related to theTRP-PLIK2, TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2d polypeptides andpolynucleotides, and therapeutic methods for treating such diseases,disorders, and/or conditions. The invention further relates to screeningmethods for identifying binding partners of the polypeptides.

[0019] The invention further provides an isolated TRP-PLIK2 polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0020] The invention further provides an isolated TRP-PLIK2b polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0021] The invention further provides an isolated TRP-PLIK2c polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0022] The invention further provides an isolated TRP-PLIK2d polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0023] The invention further relates to a polynucleotide encoding apolypeptide fragment of SEQ ID NO:2, 4, 6, 8, and/or 98, or apolypeptide fragment encoded by the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1, 3, 5, 7, and/or97.

[0024] The invention further relates to a polynucleotide encoding apolypeptide domain of SEQ ID NO:2, 4, 6, 8, and/or 98 or a polypeptidedomain encoded by the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:1, 3, 5, 7, and/or 97.

[0025] The invention further relates to a polynucleotide encoding apolypeptide epitope of SEQ ID NO:2, 4, 6, 8, and/or 98 or a polypeptideepitope encoded by the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:1, 3, 5, 7, and/or 97.

[0026] The invention further relates to a polynucleotide encoding apolypeptide of SEQ ID NO:2, 4, 6, 8, and/or 98 or the cDNA sequenceincluded in the deposited clone, which is hybridizable to SEQ ID NO:1,3, 5, 7, and/or 97, having biological activity.

[0027] The invention further relates to a polynucleotide which is avariant of SEQ ID NO:1, 3, 5, 7, and/or 97.

[0028] The invention further relates to a polynucleotide which is anallelic variant of SEQ ID NO:1, 3, 5, 7, and/or 97.

[0029] The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:2, 4, 6, 8, and/or 98.

[0030] The invention further relates to a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:1, 3, 5,7, and/or 97.

[0031] The invention further relates to a polynucleotide capable ofhybridizing under stringent conditions to any one of the polynucleotidesspecified herein, wherein said polynucleotide does not hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence of only A residues or of only T residues.

[0032] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:2, 4, 6, 8, and/or 98, wherein the polynucleotidefragment comprises a nucleotide sequence encoding an transient potentialreceptor protein.

[0033] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO: 1, 3, 5, 7, and/or 97, wherein the polynucleotidefragment comprises a nucleotide sequence encoding the sequenceidentified as SEQ ID NO:2, 4, 6, 8, and/or 98 or the polypeptide encodedby the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1, 3, 5, 7, and/or 97.

[0034] The invention further relates to an isolated nucleic acidmolecule of of SEQ ID NO:1, 3, 5, 7, and/or 97, wherein thepolynucleotide fragment comprises the entire nucleotide sequence of SEQID NO:1, 3, 5, 7, and/or 97 or the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1, 3, 5, 7, and/or97.

[0035] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:1, 3, 5, 7, and/or 97, wherein the nucleotidesequence comprises sequential nucleotide deletions from either theC-terminus or the N-terminus.

[0036] The invention further relates to an isolated polypeptidecomprising an amino acid sequence that comprises a polypeptide fragmentof SEQ ID NO:2, 4, 6, 8, and/or 98 or the encoded sequence included inthe deposited clone.

[0037] The invention further relates to a polypeptide fragment of SEQ IDNO:2, 4, 6, 8, and/or 98 or the encoded sequence included in thedeposited clone, having biological activity.

[0038] The invention further relates to a polypeptide domain of SEQ IDNO:2, 4, 6, 8, and/or 98 or the encoded sequence included in thedeposited clone.

[0039] The invention further relates to a polypeptide epitope of SEQ IDNO:2, 4, 6, 8, and/or 98 or the encoded sequence included in thedeposited clone.

[0040] The invention further relates to a full length protein of SEQ IDNO:2, 4, 6, 8, and/or 98 or the encoded sequence included in thedeposited clone.

[0041] The invention further relates to a variant of SEQ ID NO:2, 4, 6,8, and/or 98.

[0042] The invention further relates to an allelic variant of SEQ IDNO:2, 4, 6, 8, and/or 98. The invention further relates to a specieshomologue of SEQ ID NO:2, 4, 6, 8, and/or 98.

[0043] The invention further relates to the isolated polypeptide of ofSEQ ID NO:2, 4, 6, 8, and/or 98, wherein the full length proteincomprises sequential amino acid deletions from either the C-terminus orthe N-terminus.

[0044] The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:2, 4, 6, 8, and/or98.

[0045] The invention further relates to a method for preventing,treating, or ameliorating a medical condition, comprising administeringto a mammalian subject a therapeutically effective amount of thepolypeptide of SEQ ID NO:2, 4, 6, 8, and/or 98 or the polynucleotide ofSEQ ID NO:1, 3, 5, 7, and/or 97.

[0046] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence orabsence of a mutation in the polynucleotide of SEQ ID NO:1, 3, 5, 7,and/or 97; and (b) diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence orabsence of said mutation.

[0047] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence oramount of expression of the polypeptide of of SEQ ID NO:2, 4, 6, 8,and/or 98 in a biological sample; and diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thepresence or amount of expression of the polypeptide.

[0048] The invention further relates to a method for identifying abinding partner to the polypeptide of SEQ ID NO:2, 4, 6, 8, and/or 98comprising the steps of (a) contacting the polypeptide of SEQ ID NO:2,4, 6, 8, and/or 98 with a binding partner; and (b) determining whetherthe binding partner effects an activity of the polypeptide.

[0049] The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:1, 3, 5, 7, and/or 97.

[0050] The invention further relates to a method of identifying anactivity in a biological assay, wherein the method comprises the stepsof expressing SEQ ID NO:1, 3, 5, 7, and/or 97 in a cell, (b) isolatingthe supernatant; (c) detecting an activity in a biological assay; and(d) identifying the protein in the supernatant having the activity.

[0051] The invention further relates to a process for makingpolynucleotide sequences encoding gene products having altered SEQ IDNO:2, 4, 6, 8, and/or 98 activity comprising the steps of (a) shufflinga nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, and/or 97, (b)expressing the resulting shuffled nucleotide sequences and, (c)selecting for altered activity as compared to the activity of the geneproduct of said unmodified nucleotide sequence.

[0052] The invention further relates to a shuffled polynucleotidesequence produced by a shuffling process, wherein said shuffled DNAmolecule encodes a gene product having enhanced tolerance to aninhibitor of SEQ ID NO:2, 4, 6, 8, and/or 98 activity.

[0053] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is agastrointestinal disorder.

[0054] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is a metabolicdisorder.

[0055] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is an immunedisorder

[0056] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is a hematopoieticdisorder.

[0057] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is a inflammatorydisorder.

[0058] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is a renaldisorder.

[0059] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is a reproductivedisorder.

[0060] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is a hepaticdisorder.

[0061] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is adisorderrelated to hyper transient receptor potential activity.

[0062] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is prostate cancer.

[0063] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptideprovided as SEQ ID NO:2, 4, 6, 8, and/or 98, in addition to, itsencoding nucleic acid, wherein the medical condition is amyotrophiclateral sclerosis with frontotemporal dementia, early-onset pulverulentcataract, infantile nephronophthisis, hypomagnesemia with secondaryhypocalcemia, familial hemophagocytic lymphohistiocytosis, neuronaldegeneration, neurogenic inflammation, allergy,immunodeficiency/excessive immune activation, visual defects, hearingdisorder, pain, cancer, hypertension, other cardiovascular diseases,diseases associated with disturbances in Ca²⁺ homeostasis includingosteoporosis, hypercalciuric stone disease, and chronic renal failure

[0064] The invention further relates to a method of identifying acompound that modulates the biological activity of TRP-PLIK2, comprisingthe steps of, (a) combining a candidate modulator compound withTRP-PLIK2 having the sequence set forth in one or more of SEQ ID NO:2,4, 6, 8, and/or 98; and measuring an effect of the candidate modulatorcompound on the activity of TRP-PLIK2.

[0065] The invention further relates to a method of identifying acompound that modulates the biological activity of a transient receptorpotential protein, comprising the steps of, (a) combining a candidatemodulator compound with a host cell expressing TRP-PLIK2 having thesequence as set forth in SEQ ID NO:2, 4, 6, 8, and/or 98; and, (b)measuring an effect of the candidate modulator compound on the activityof the expressed TRP-PLIK2.

[0066] The invention further relates to a method of identifying acompound that modulates the biological activity of TRP-PLIK2, comprisingthe steps of, (a) combining a candidate modulator compound with a hostcell containing a vector described herein, wherein TRP-PLIK2 isexpressed by the cell; and, (b) measuring an effect of the candidatemodulator compound on the activity of the expressed TRP-PLIK2.

[0067] The invention further relates to a method of screening for acompound that is capable of modulating the biological activity ofTRP-PLIK2, comprising the steps of: (a) providing a host cell describedherein; (b) determining the biological activity of TRP-PLIK2 in theabsence of a modulator compound; (c) contacting the cell with themodulator compound; and (d)determining the biological activity ofTRP-PLIK2 in the presence of the modulator compound; wherein adifference between the activity of TRP-PLIK2 in the presence of themodulator compound and in the absence of the modulator compoundindicates a modulating effect of the compound.

[0068] The invention further relates to a compound that modulates thebiological activity of human TRP-PLIK2 as identified by the methodsdescribed herein.

[0069] The present invention also relates to an isolated polynucleotideconsisting of a portion of the human TRP-PLIK2 gene consisting of atleast 8 bases, specifically excluding Genbank Accession Nos. B1754451,BB593693, BM472126, BE465471, AW645658, BB650718, A1390333, AW916907,BJ028793, and/or BE305668.

[0070] The present invention also relates to an isolated polynucleotideconsisting of a nucleotide sequence encoding a fragment of the humanTRP-PLIK2 protein, wherein said fragment displays one or more functionalactivities specifically excluding Genbank Accession Nos. B1754451,BB593693, BM472126, BE465471, AW645658, BB650718, A1390333, AW916907,BJ028793, and/or BE305668.

[0071] The present invention also relates to the polynucleotide of SEQID NO:1, 3, 5, 7, and/or 97 consisting of at least 10 to 50 bases,wherein said at least 10 to 50 bases specifically exclude thepolynucleotide sequence of Genbank Accession Nos. B1754451, BB593693,BM472126, BE465471, AW645658, BB650718, A1390333, AW916907, BJ028793,and/or BE305668.

[0072] The present invention also relates to the polynucleotide of SEQID NO:1, 3, 5, 7, and/or 97 consisting of at least 15 to 100 bases,wherein said at least 15 to 100 bases specifically exclude thepolynucleotide sequence of Genbank Accession Nos. B1754451, BB593693,BM472126, BE465471, AW645658, BB650718, A1390333, AW916907, BJ028793,and/or BE305668.

[0073] The present invention also relates to the polynucleotide of SEQID NO:1, 3, 5, 7, and/or 97 consisting of at least 100 to 1000 bases,wherein said at least 100 to 1000 bases specifically exclude thepolynucleotide sequence of Genbank Accession Nos. B1754451, BB593693,BM472126, BE465471, AW645658, BB650718, A1390333, AW916907, BJ028793,and/or BE305668.

[0074] The present invention also relates to an isolated polypeptidefragment of the human TRP-PLIK2 protein, wherein said polypeptidefragment does not consist of the polypeptide encoded by thepolynucleotide sequence of Genbank Accession Nos. B1754451, BB593693,BM472126, BE465471, AW645658, BB650718, A1390333, AW916907, BJ028793,and/or BE305668.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

[0075] FIGS. 1A-G show the polynucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of the novel human transientreceptor potential channel member, TRP-PLIK2, of the present invention.The standard one-letter abbreviation for amino acids is used toillustrate the deduced amino acid sequence. The polynucleotide sequencecontains a sequence of 6054 nucleotides (SEQ ID NO:1), encoding apolypeptide of 2017 amino acids (SEQ ID NO:2). An analysis of theTRP-PLIK2 polypeptide determined that it comprised the followingfeatures: six transmembrane domains (TM1 thru TM6) located from aboutamino acid 740 to about amino acid 757 (TM1), from about amino acid 834to about amino acid 851 (TM2), from about amino acid 908 to about aminoacid 920 (TM3), from about amino acid 934 to about amino acid 951 (TM4),from about amino acid 968 to about amino acid 985 (TM5), and/or fromabout amino acid 1043 to about amino acid 1062 (TM6) of SEQ ID NO:2represented by double underlining; a predicted TRP domain (LWKYNR)located from about amino acid 1078 to about amino acid 1083 of SEQ IDNO:2 represented by light shading; a predicted nucleotide binding domainlocated from about amino acid 1945 to about amino acid 1950 of SEQ IDNO:2 represented by dark shading; a predicted zinc finger domain locatedat about amino acid 1960 to about amino acid 1970 of SEQ ID NO:2represented by dotted underlining; a predicted ion transport signaturedomain located at about amino acid 904 to about amino acid 1064 of SEQID NO:2 represented by italics; conserved cysteine residues located atamino acid 21, 34, 38, 41, 47, 49, 311, 367, 637, 702, 719, 938, 1028,1034, 1114, 1148, 1822, 1861, 1963, 1966, and 1967 of SEQ ID NO:2represented in bold. Conservation of cysteines at key amino acidresidues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0076] FIGS. 2A-G show the polynucleotide sequence (SEQ ID NO:3) anddeduced amino acid sequence (SEQ ID NO:4) of the novel human transientreceptor potential channel member splice variant, TRP-PLIK2b, of thepresent invention. The standard one-letter abbreviation for amino acidsis used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 5913 nucleotides (SEQ IDNO:3), encoding a polypeptide of 1970 amino acids (SEQ ID NO:4). Ananalysis of the TRP-PLIK2b polypeptide determined that it comprised thefollowing features: six transmembrane domains (TM1 thru TM6) locatedfrom about amino acid 693 to about amino acid 710 (TM1), from aboutamino acid 787 to about amino acid 804 (TM2), from about amino acid 861to about amino acid 873 (TM3), from about amino acid 887 to about aminoacid 904 (TM4), from about amino acid 921 to about amino acid 938 (TM5),and/or from about amino acid 996 to about amino acid 1015 (TM6) of SEQID NO:4 represented by double underlining; a predicted TRP domain(LWKYNR) located from about amino acid 1031 to about amino acid 1036 ofSEQ ID NO:4 represented by light shading; a predicted nucleotide bindingdomain located from about amino acid 1898 to about amino acid 1903 ofSEQ ID NO:4 represented by dark shading; a predicted zinc finger domainlocated at about amino acid 1913 to about amino acid 1923 of SEQ ID NO:2represented by dotted underlining; a predicted ion transport signaturedomain located at about amino acid 857 to about amino acid 1017 of SEQID NO:4 represented by italics; conserved cysteine residues located atamino acid 21, 34, 38, 41, 47, 49, 311, 367, 590, 655, 672, 891, 981,987, 1067, 1101, 1775, 1814, 1915, 1919, and 1920 of SEQ ID NO:4represented in bold. Conservation of cysteines at key amino acidresidues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0077] FIGS. 3A-G show the polynucleotide sequence (SEQ ID NO:5) anddeduced amino acid sequence (SEQ ID NO:6) of the novel human transientreceptor potential channel member splice variant, TRP-PLIK2c, of thepresent invention. The standard one-letter abbreviation for amino acidsis used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 5820 nucleotides (SEQ IDNO:5), encoding a polypeptide of 1939 amino acids (SEQ ID NO:6). Ananalysis of the TRP-PLIK2c polypeptide determined that it comprised thefollowing features: six transmembrane domains (TM1 thru TM6) locatedfrom about amino acid 662 to about amino acid 679 (TM1), from aboutamino acid 756 to about amino acid 773 (TM2), from about amino acid 830to about amino acid 842 (TM3), from about amino acid 856 to about aminoacid 873 (TM4), from about amino acid 890 to about amino acid 907 (TM5),and/or from about amino acid 965 to about amino acid 984 (TM6) of SEQ IDNO:6 represented by double underlining; a predicted TRP domain (LWKYNR)located from about amino acid 1000 to about amino acid 1005 of SEQ IDNO:6 represented by light shading; a predicted nucleotide binding domainlocated from about amino acid 1867 to about amino acid 1872 of SEQ IDNO:6 represented by dark shading; a predicted zinc finger domain locatedat about amino acid 1882 to about amino acid 1892 of SEQ ID NO:2represented by dotted underlining; a predicted ion transport signaturedomain located at about amino acid 826 to about amino acid 986 of SEQ IDNO:6 represented by italics; conserved cysteine residues located atamino acid 21, 34, 38, 41, 47, 49, 311, 367, 590, 655, 672, 891, 981,987, 1067, 1101, 1775, 1814, 1915, 1919, and 1920 of SEQ ID NO:6represented in bold. Conservation of cysteines at key amino acidresidues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0078] FIGS. 4A-G show the polynucleotide sequence (SEQ ID NO:7) anddeduced amino acid sequence (SEQ ID NO:8) of the novel human transientreceptor potential channel member splice variant, TRP-PLIK2d, of thepresent invention. The standard one-letter abbreviation for amino acidsis used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 5925 nucleotides (SEQ IDNO:7), encoding a polypeptide of 1974 amino acids (SEQ ID NO:8). Ananalysis of the TRP-PLIK2d polypeptide determined that it comprised thefollowing features: six transmembrane domains (TM1 thru TM6) locatedfrom about amino acid 740 to about amino acid 757 (TMI), from aboutamino acid 834 to about amino acid 845 (TM2), from about amino acid 861to about amino acid 880 (TM3), from about amino acid 892 to about aminoacid 909 (TM4), from about amino acid 925 to about amino acid 946 (TM5),and/or from about amino acid 996 to about amino acid 1026 (TM6) of SEQID NO:8 represented by double underlining; a predicted TRP domain(LWKYNR) located from about amino acid 1035 to about amino acid 1040 ofSEQ ID NO:8 represented by light shading; a predicted nucleotide bindingdomain located from about amino acid 1902 to about amino acid 1907 ofSEQ ID NO:8 represented by dark shading; a predicted zinc finger domainlocated at about amino acid 1917 to about amino acid 1927 of SEQ ID NO:2represented by dotted underlining; a predicted ion transport signaturedomain located at about amino acid 904 to about amino acid 959 of SEQ IDNO:8 represented by italics; conserved cysteine residues located atamino acid 21, 34, 38, 41, 47, 49, 311, 367, 637, 702, 719, 895. 985,991, 1071, 1105, 1779, 1818, 1920, 1923, and 1924 of SEQ ID NO:8represented in bold. Conservation of cysteines at key amino acidresidues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0079] FIGS. 5A-F show the regions of identity and similarity betweenthe TRP-PLIK2, TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2d polypeptides ofthe present invention to another member of human transient receptorpotential channel family, specifically, the human channel-kinase 1protein, also known as the human CHAK1 or TRP-PLIK1 protein (CHAKI;Genbank Accession No. gilAF346629; SEQ ID NO:9); and the humanmelastatin 1 protein (Melastatinl; Genbank Accession No. gil3243075; SEQID NO:260). The alignment was created using the CLUSTALW algorithmdescribed elsewhere herein using default parameters (CLUSTALWparameters: gap opening penalty: 10; gap extension penalty: 0.5; gapseparation penalty range: 8; percent identity for alignment delay: 40%;and transition, weighting: 0). The darkly shaded amino acids representregions of matching identity. The lightly shaded amino acids representregions of matching similarity. Dots between residues indicate gappedregions for the aligned polypeptides.

[0080]FIG. 6A-E shows the regions of identity between the TRP-PLIK2polypeptide (SEQ ID NO:2) of the present invention to its predictedsplice variants TRP-PLIK2b (SEQ ID NO:4), TRP-PLIK2c (SEQ ID NO:6), andTRP-PLIK2d (SEQ ID NO:8) polypeptides of the present invention. Thealignment was created using the CLUSTALW algorithm described elsewhereherein using default parameters (CLUSTALW parameters: gap openingpenalty: 10; gap extension penalty: 0.5; gap separation penalty range:8; percent identity for alignment delay: 40%; and transition, weighting:0). The darkly shaded amino acids represent regions of matchingidentity. The lightly shaded amino acids represent regions of matchingsimilarity. Dots between residues indicate gapped regions for thealigned polypeptides.

[0081]FIG. 7 shows an expression profile of the novel human transientreceptor potential channel family member, TRP-PLIK2 (SEQ ID NO:2). Thefigure illustrates the relative expression level of TRP-PLIK2 amongstvarious mRNA tissue sources. As shown, transcripts corresponding toTRP-PLIK2 expressed predominately in bone marrow, kideny, and testistissue. The TRP-PLIK2 polypeptide was also expressed significantly inliver, and to a lesser extent, in small intestine, spinal cord,prostate, uterus, lung, lymph node, stomach, heart, brain, thymus, andpancrase. Expression data was obtained by measuring the steady stateTRP-PLIK2 mRNA levels by RT-PCR using the PCR primer pair provided asSEQ ID NO:27 and 28 as described herein.

[0082]FIG. 8 shows an expression profile of the novel human transientreceptor potential channel family member, TRP-PLIK2 (SEQ ID NO:2). Thefigure illustrates the relative expression level of TRP-PLIK2 amongstvarious mRNA tissue, and cell sources. As shown, transcriptscorresponding to TRP-PLIK2 expressed predominately in kidney tissue. TheTRP-PLIK2 polypeptide was also expressed significantly in brain, andskeletal muscle. Expression data was obtained by probing a Northern blotusing a TRP-PLIK2 507-bp PCR amplified fragment as described herein.

[0083]FIG. 9 shows a table illustrating the percent identity and percentsimilarity between the TRP-PLIK2, TRP-PLIK2b, TRP-PLIK2c, and TRP-PLIK2dpolypeptides of the present invention with the human CHAK1 protein(CHAKI; Genbank Accession No. gilAF346629; SEQ ID NO:9) ; and the humanmelastatin 1 protein (Melastatinl; Genbank Accession No. gil3243075; SEQID NO:260). The percent identity and percent similarity values weredetermined based upon the GAP algorithm (GCG suite of programs; andHenikoff, S. and Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89:10915-10919(1992)).

[0084] FIGS. 10A-B shows the polynucleotide sequences from the human bacAL354795 used to design primers for cloning the TRP-PLIK2, TRP-PLIK2b,TRP-PLIK2c, and TRP-PLIK2d polynucleotides of the present invention asdescribed herein.

[0085]FIG. 11A-C show a partial polynucleotide sequence (SEQ ID NO:97)and partial deduced amino acid sequence (SEQ ID NO:98) of the novelhuman transient receptor potential channel member, TRP-PLIK2, of thepresent invention. The standard one-letter abbreviation for amino acidsis used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 1933 nucleotides (SEQ IDNO:97), encoding a polypeptide of 496 amino acids (SEQ ID NO:98).

[0086]FIG. 12 shows an expanded expression profile of the novel humantransient receptor potential channel member, TRP-PLIK2. The figureillustrates the relative expression level of TRP-PLIK2 amongst variousmRNA tissue sources. As shown, the TRP-PLIK2 polypeptide was expressedpredominately in the lower gastrointestinal tract, specifically theileum, the rectum, the colon, the jejunum, duodenum, with minortranscript levels observed in the stomach. Expression of TRP-PLIK2 wasalso significantly expressed in the kidney, particularly in the cortex,followed by the medulla, and to a lesser extent in the testis.Expression data was obtained by measuring the steady state TRP-PLIK2mRNA levels by quantitative PCR using the PCR primer pair provided asSEQ ID NO:267 and 268, and Taqman probe (SEQ ID NO:269) as described inExample 4 herein.

[0087]FIG. 13 shows the results of a kinase assay for the TRP-PLIK2-GSTfusion protein polypeptide designed to measure the level of incorporatedp³² in either MBP or Histon protein substrates. GST fusion vectorlacking the TRP-PLIK2 kinase domain was used as a control (“Vector”).The TRP-PLIK2-GST fusion protein is represented as “GST-PLIK2 kinase”,the TRP-PLIK2 kinase domain is represented as “PLIK2 kinase”, and “MBP”represents the MBP protein. As shown, predicted TRP-PLIK2 kinase domainwas determined to have kinase activity as demonstrated by thephosphorylated MBP protein. Moreover, TRP-PLIK2 was also determined tobe capable of autophosphorylation as demonstrated by the intensephosphorylation of the TRP-PLIK2 kinase domain (“PLIK2 kinase”). TheTRP-PLIK2-GST fusion protein contained only the predicted kinase domainof the TRP-PLIK2 polypeptide as described in Example 5 herein.

[0088]FIG. 14 shows an expanded expression profile of the novel humantransient receptor potential channel member, TRP-PLIK2. The figureillustrates the relative expression level of TRP-PLIK2 amongst variousnormal and tumor mRNA tissue sources. As shown, the TRP-PLIK2polypeptide was differentially expressed to the greatest extent inprostate tumor tissue relative to normal prostate tissue (approximately20 fold difference). Expression of TRP-PLIK2 was also significantlydifferentially expressed in the testicular tumors relative to normaltesticular tissue. Expression data was obtained by measuring the steadystate TRP-PLIK2 mRNA levels by quantitative PCR using the PCR primerpair provided as SEQ ID NO:267 and 268, and Taqman probe (SEQ ID NO:269)as described in Example 4 herein.

[0089]FIG. 15 shows the results of an immunoprecipitation assay for theHA tagged TRP-PLIK2-GST fusion protein polypeptide designed to proveTRP-PLIK2 is expressed in HEK cells. GST fusion vector lacking theencoding TRP-PLIK2 polynucleotide sequence was used as a control(“Vector”). The HA tagged TRP-PLIK2-GST fusion protein is represented as“LTRPC6”. The immunoprecipitation experiments were performed asdescribed in Example 5 herein.

[0090] Table I provides a summary of the novel polypeptides and theirencoding polynucleotides of the present invention.

[0091] Table II illustrates the preferred hybridization conditions forthe polynucleotides of the present invention. Other hybridizationconditions may be known in the art or are described elsewhere herein.

[0092] Table III provides a summary of various conservativesubstitutions encompassed by the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included herein. All references to“TRP-PLIK2“shall be construed to apply to “TRP-PLIK2”, “TRP-PLIK2b”,“TRP-PLIK2c”, and/or “TRP-PLIK2d” unless otherwise specified herein.

[0094] The invention provides a novel human sequence that potentiallyencodes a novel human transient receptor potential channel family membercalled TRP-PLIK2, in addition to, its splice variants TRP-PLIK2b,TRP-PLIK2c, and TRP-PLIK2d. TRP-PLIK2 shares significant homologue withother transient receptor potential channel family members, such as humanCHAKI. Transcripts for TRP-PLIK2 were found predominately in the kidney,gastrointestinal tract, bone marrow, and testis suggesting that theinvention potentially modulates leukocyte proliferation,differentiation, migration, and activation in these tissues. Therefore,the polynucleotide of the present invention has been tentatively namedTRP-PLIK2.

[0095] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide. The term “isolated” does not refer to genomic or cDNAlibraries, whole cell total or mRNA preparations, genomic DNApreparations (including those separated by electrophoresis andtransferred onto blots), sheared whole cell genomic DNA preparations orother compositions where the art demonstrates no distinguishing featuresof the polynucleotide/sequences of the present invention.

[0096] In specific embodiments, the polynucleotides of the invention areat least 15, at least 30, at least 50, at least 100, at least 125, atleast 500, or at least 1000 continuous nucleotides but are less than orequal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotidesof the invention comprise a portion of the coding sequences, asdisclosed herein, but do not comprise all or a portion of any intron. Inanother embodiment, the polynucleotides comprising coding sequences donot contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′to the gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

[0097] As used herein, a “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:I, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:97 or the cDNA contained within the clonedeposited with the ATCC. For example, the polynucleotide can contain thenucleotide sequence of the full length cDNA sequence, including the 5′and 3′ untranslated sequences, the coding region, with or without asignal sequence, the secreted protein coding region, as well asfragments, epitopes, domains, and variants of the nucleic acid sequence.Moreover, as used herein, a “polypeptide” refers to a molecule havingthe translated amino acid sequence generated from the polynucleotide asbroadly defined.

[0098] In the present invention, the full length sequence identified asSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and, SEQ ID NO:97 wasoften generated by overlapping sequences contained in multiple clones(contig analysis). A representative clone containing all or most of thesequence for SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQID NO:97 was deposited with the American Type Culture Collection(“ATCC”). As shown in Table 1, each clone is identified by a cDNA CloneID (Identifier) and the ATCC Deposit Number. The ATCC is located at10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCCdeposit was made pursuant to the terms of the Budapest Treaty on theinternational recognition of the deposit of microorganisms for purposesof patent procedure. The deposited clone is inserted in the pCR4Blunt-TOPO plasmid (Invitrogen) as described herein.

[0099] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequnencer (such as the Model 373, preferably a Model 3700, fromApplied Biosystems, Inc.), and all amino acid sequences of polypeptidesencoded by DNA molecules determined herein were pridcted by translationof a DNA sequence determined above. Therefore, as is known in the artfor any DNA seuqnece detemrined by this automated approach, anynucleotide seqence determined herein may contain some errors. Nucleotidesequences determined by automation are typically at least about 90%identical, more typically at least about 95% to at least about 99.9%identical to the actual nucleotide seqnece of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart. As is also known in the art, a single insertion or deletion in adetemrined nucleotide sequence compared to the actual sequence willcause a frame shift in translation of the nucleotide sequence such thatthe predicted amino acid sequence encoded by a determined nucleotidesequence will be completely different from the amino acid sequenceactually encoded bt the sequenced DNA molecule, beginning at the pointof such an insertion or deletion.

[0100] Using the information provided herein, such as the nucleotidesequence in FIGS. 1A-G (SEQ ID NO:1), a nucleic acid molecule of thepresent invention encoding the TRP-PLIK2 polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in FIGS. 1A-G (SEQ IDNO:l) was discovered in a cDNA library derived from brain, fetal brain,heart, fetal heart, kidney and fetal kidney.

[0101] Using the information provided herein, such as the nucleotidesequence in FIGS. 2A-G (SEQ ID NO:3), a nucleic acid molecule of thepresent invention encoding the TRP-PLIK2b polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in FIGS. 2A-G (SEQ IDNO:3) was discovered in a cDNA library derived from brain, fetal brain,heart, fetal heart, kidney and fetal kidney.

[0102] Using the information provided herein, such as the nucleotidesequence in FIGS. 3A-G (SEQ ID NO:5), a nucleic acid molecule of thepresent invention encoding the TRP-PLIK2c polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in FIGS. 3A-G (SEQ IDNO:5) was discovered in a cDNA library derived from brain, fetal brain,heart, fetal heart, kidney and fetal kidney.

[0103] Using the information provided herein, such as the nucleotidesequence in FIGS. 4A-G (SEQ ID NO:7), a nucleic acid molecule of thepresent invention encoding the TRP-PLIK2d polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in FIGS. 4A-G (SEQ IDNO:7) was discovered in a cDNA library derived from brain, fetal brain,heart, fetal heart, kidney and fetal kidney.

[0104] A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:I, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:97, the complements thereof, topolynucleotide sequences encoding the sequences contained in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:98, thecomplements thereof, or the cDNA within the clone deposited with theATCC. “Stringent hybridization conditions” refers to an overnightincubation at 42 degree C. in a solution comprising 50% formamide, 5xSSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.lx SSC at about 65 degree C.

[0105] Also contemplated are nucleic acid molecules that hybridize tothe polynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency), salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

[0106] Note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

[0107] Of course, a polynucleotide which hybridizes only topolyA+sequences (such as any 3′ terminal polyA+tract of a cDNA shown inthe sequence listing), or to a complementary stretch of T (or U)residues, would not be included in the definition of “polynucleotide,”since such a polynucleotide would hybridize to any nucleic acid moleculecontaining a poly (A) stretch or the complement thereof (e.g.,practically any double-stranded cDNA clone generated using oligo dT as aprimer).

[0108] The polynucleotide of the present invention can be composed ofany polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

[0109] The polypeptide of the present invention can be composed of aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

[0110] “SEQ ID NO:X” refer to polynucleotide sequences, while “SEQ IDNO:Y” refer to polypeptide sequences, all sequences being identified byan integer specified in Table I herein.

[0111] “A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

[0112] The term “organism” as referred to herein is meant to encompassany organism referenced herein, though preferably to eukaryoticorganisms, more preferably to mammals, and most preferably to humans.

[0113] The present invention encompasses the identification of proteins,nucleic acids, or other molecules, that bind to polypeptides andpolynucleotides of the present invention (for example, in areceptor-ligand interaction). The polynucleotides of the presentinvention can also be used in interaction trap assays (such as, forexample, that discribed by Ozenberger and Young (Mol Endocrinol.,9(10):1321-9, (1995); and Ann. N. Y. Acad. Sci., 7;766:279-81, (1995)).

[0114] The polynucleotide and polypeptides of the present invention areuseful as probes for the identification and isolation of full-lengthcDNAs and/or genomic DNA which correspond to the polynucleotides of thepresent invention, as probes to hybridize and discover novel, relatedDNA sequences, as probes for positional cloning of this or a relatedsequence, as probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides, as probes to quantify geneexpression, and as probes for microarays.

[0115] In addition, polynucleotides and polypeptides of the presentinvention may comprise one, two, three, four, five, six, seven, eight,or more membrane domains.

[0116] Also, in preferred embodiments the present invention providesmethods for further refining the biological fuction of thepolynucleotides and/or polypeptides of the present invention.

[0117] Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

[0118] In preferred embodiments, the invention provides methods foridentifying the glycosylation sites inherent in the polynucleotides andpolypeptides of the invention, and the subsequent alteration, deletion,and/or addition of said sites for a number of desirable characteristicswhich include, but are not limited to, augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

[0119] In further preferred embodiments, methods are provided forevolving the polynucleotides and polypeptides of the present inventionusing molecular evolution techniques in an effort to create and identifynovel variants with desired structural, functional, and/or physicalcharacteristics.

[0120] The present invention further provides for other experimentalmethods and procedures currently available to derive functionalassignments. These procedures include but are not limited to spotting ofclones on arrays, micro-array technology, PCR based methods (e.g.,quantitative PCR), anti-sense methodology, gene knockout experiments,and other procedures that could use sequence information from clones tobuild a primer or a hybrid partner.

[0121] As used herein the terms “modulate” or “modulates” refer to anincrease or decrease in the amount, quality or effect of a particularactivity, DNA, RNA, or protein.

[0122] Polynucleotides and Polypeptides of the Invention

[0123] Features of the Polypeptide Encoded by Gene No:1

[0124] The polypeptide of this gene provided as SEQ ID NO:2 (FIGS.1A-G), encoded by the polynucleotide sequence according to SEQ ID NO:1(FIGS. 1A-G), and/or encoded by the polynucleotide contained within thedeposited clone, TRP-PLIK2, has significant homology at the nucleotideand amino acid level to the human channel-kinase 1 protein, also knownas the human CHAK1 or TRP-PLIK1 protein (CHAKI; Genbank Accession No.gilAF346629; SEQ ID NO:9) ; and the human melastatin 1 protein(Melastatinl; Genbank Accession No. gil3243075; SEQ ID NO:260). Analignment of the TRP-PLIK2 polypeptide with this protein is provided inFIGS. 5A-F.

[0125] The TRP-PLIK2 polypeptide was determined to share 58.0% identityand 66.0% similarity with the human CHAK1 or TRP-PLIK1 protein (CHAKI;Genbank Accession No. gilAF346629; SEQ ID NO:9); and was determined toshare 48.1% identity and 58.6% similarity with the human melastatin 1protein (Melastatinl; Genbank Accession No. gil3243075; SEQ ID NO:260)as shown in FIG. 9.

[0126] The CHAK1 protein is believed to represent a member of a newclass of protein kianses referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain.

[0127] The melastatin 1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin 1 could represent a noveltherapeutic in the treatment of melanoa and potentially other cancers.

[0128] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the CHAK1 and melastatin 1 proteins, in addition to,other transient receptor potential channel family members referencedelsewhere herein or otherwise known in the art.

[0129] Consistent with the TRP-PLIK2 polypeptide representing a noveltransient receptor potential channel family member, the predictedTRP-PLIK2 kinase domain polypeptide was determined to possess kinaseactivity as shown in FIG. 13 and described in Example 5. Demonstrationof kinase activity was determined by creating and purifying a TRP-PLIK2kinase domain/GST fusion protein, and subjecting it to a kinase assay.The results also demonstrated that the TRP-PLIK2-GST fusion proteincould be autophosphorylated and phosphorylate substrate polypeptides.MBP was determined to represent a preferred substrate over Histon, asdescribed in Example 5. Evidence of autophosphorylation activity isdemonstrated by the intense phosphorylation of the PLIK2 kinase domainband (“PLIK2 kinase”), in addition to the phosphorylation of theTRP-PLIK2-GST fusion protein band (“GST-PLIK2 kinase”).

[0130] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theTRP-PLIK2 polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 1A-G. The transmembrane domains arelocated from about amino acid 740 to about amino acid 757 (TM1), fromabout amino acid 834 to about amino acid 851 (TM2), from about aminoacid 908 to about amino acid 920 (TM3), from about amino acid 934 toabout amino acid 951 (TM4), from about amino acid 968 to about aminoacid 985 (TM5), and/or from about amino acid 1043 to about amino acid1062 (TM6) of SEQ ID NO:2. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0131] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKIIISIILPPTILTLEF (SEQ ID NO:45), IVKFWFYTMAYLAFLMLF (SEQ ID NO:46),TETVAIGLFSAGF (SEQ ID NO:47), RLIYCIDIIFWFSRLLDF (SEQ ID NO:48),MTANMFYIVIIMAIVLLS (SEQ ID NO:49), and/or FLQAVYLFVQYIIMVNLLIA (SEQ IDNO:50). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the TRP-PLIK2transmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0132] In preferred embodiments, the present invention encompasses theuse of N-terminal deletions, C-terminal deletions, or any combination ofN-terminal and C-terminal deletions of any one or more of the TRP-PLIK2TM1 thru TM6 transmembrane domain polypeptides as antigenic and/orimmunogenic epitopes.

[0133] In preferred embodiments, the present invention also encompassesthe use of N-terminal deletions, C-terminal deletions, or anycombination of N-terminal and C-terminal deletions of any one or more ofthe amino acids intervening (i.e., ion channel extracellular orintracellular loops) the TRP-PLIK2 TM1 thru TM6 transmembrane domainpolypeptides as antigenic and/or immunogenic epitopes.

[0134] The TRP-PLIK2 polypeptide was determined to comprise severalconserved cysteines, at amino acid 21, 34, 38, 41, 47, 49, 311, 367,637, 702, 719, 938, 1028, 1034, 1114, 1148, 1822, 1861, 1963, 1966, and1967 of SEQ ID No: 2 (FIGS. 1A-D). Conservation of cysteines at keyamino acid residues is indicative of conserved structural features,which may correlate with conservation of protein function and/oractivity.

[0135] In confirmation of the TRP-PLIK2 representing a member of thetransient receptor channel family, the TRP-PLIK2 polypeptide wasdetermined to comprise a predicted TRP domain (LWKYNR) located fromabout amino acid 1078 to about amino acid 1083 of SEQ ID NO:2. In thiscontext, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of theabove referenced polypeptide.

[0136] In further confirmation of the TRP-PLIK2 representing a member ofthe transient receptor channel family, the TRP-PLIK2 polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 904 to about amino acid 1064 of SEQ ID NO:2.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0137] The TRP-PLIK2 polypeptide was determined to comprise a predictednucleotide binding domain located from about amino acid 1945 to aboutamino acid 1950 of SEQ ID NO:2. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0138] In addition, the TRP-PLIK2 polypeptide was determined to comprisea predicted zinc finger domain located at about amino acid 1960 to aboutamino acid 1970 of SEQ ID NO:2. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0139] TRP-PLIK2 polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofTRP-PLIK2 by identifying mutations in the TRP-PLIK2 gene using TRP-PLIK2sequences as probes or by determining TRP-PLIK2 protein or mRNAexpression levels. TRP-PLIK2 polypeptides will be useful in screens forcompounds that affect the activity of the protein. TRP-PLIK2 peptidescan also be used for the generation of specific antibodies and as baitin yeast two hybrid screens to find proteins the specifically interactwith TRP-PLIK2.

[0140] Expression profiling designed to measure the steady state mRNAlevels encoding the TRP-PLIK2 polypeptide showed predominately highexpression levels in bone marrow, kidney, and testis. The TRP-PLIK2polypeptide was also significantly expressioned in liver, and to alesser extent, in small intestine, spinal cord, prostate, uterus, lung,lymph node, stomach, heart, brain, thymus, and pancrease (as shown inFIG. 7).

[0141] Expanded analysis of TRP-PLIK2 expression levels by TaqMan™quantitative PCR (see FIG. 12) confirmed that the TRP-PLIK2 polypeptideis expressed in kidney, colon, and testis (FIG. 7). TRP-PLIK2 mRNA wasexpressed predominately in the lower gastrointestinal tract,specifically the ileum, the rectum, the colon, the jejunum, and to alesser extent in the duodenum and stomach. Significant expression wasobserved in the kidney, particularly in the cortex, followed by themedulla, and to a lesser extent in the testis, pelvis, and bone marrow(mononuclear cells).

[0142] Furthermore, an expanded analysis of TRP-PLIK2 expression levelsin various tumor and normal tissues by TaqMan™ quantitative PCR (seeFIG. 14) showed TRP-PLIK2 mRNA was differentially expressed to thegreatest extent in prostate tumor tissue relative to normal prostatetissue (approximately 20 fold difference). Significant differentalexpression was also observed in the testicular tumor tissue relative tonormal testicular tissue.

[0143] Characterization of the TRP-PLIK2 polypeptide of the presentinvention using antisense oligonucleotides directed against a portion ofthe TRP-PLIK2 encoding sequence led to the determination that it isinvolved in the modulation of the NFkB pathway, either directly orindirectly.

[0144] The upregulation of IkBa due to the downregulation of TRP-PLIK2places this transient receptor potential protein into a signallingpathway potentially involved in apoptotic events. This gives theopportunity to regulate downstream events via the activity of theprotein TRP-PLIK2 with antisense polynucleotides, polypeptides or lowmolecular chemicals with the potential of achieving a therapeutic effectin cancer, autoimmune diseases. In addition to cancer and immunologicaldisorders, NF-kB has significant roles in other diseases (Baldwin, A.S., J. Clin Invest. 107, :3-6 (2001)). NF-kB is a key factor in thepathophysiology of ischemia-reperfusion injury and heart failure (Valen,G., Yan. Z Q, Hansson G K, J. Am. Coll. Cardiol. 38, 307-14 (2001)).Furthermore, NF-kB has been found to be activated in experimental renaldisease (Guijarro C, Egido J., Kidney Int. 59, 415-425 (2001)). AsTRP-PLIK2 is highly expressed in kidney there is the potential of aninvolvement in renal diseases.

[0145] In preferred embodiments, TRP-PLIK2 polynucleotides andpolypeptides, including fragments thereof, are useful for treating,diagnosing, and/or ameliorating proliferative disorders, cancers,ischemia-reperfusion injury, heart failure, immuno compromisedconditions, HIV infection, and renal diseases.

[0146] Moreover, TRP-PLIK2 polynucleotides and polypeptides, includingfragments thereof, are useful for increasing NF-kB activity, increasingapoptotic events, and/or decreasing IkBa expression or activity levels.

[0147] In preferred embodiments, antagonists directed against TRP-PLIK2are useful for treating, diagnosing, and/or ameliorating autoimmunedisorders, disorders related to hyper immune activity, inflammatoryconditions, disorders related to aberrant acute phase responses,hypercongenital conditions, birth defects, necrotic lesions, wounds,organ transplant rejection, conditions related to organ transplantrejection, disorders related to aberrant signal transduction,proliferating disorders, cancers, HIV, and HIV propagation in cellsinfected with other viruses.

[0148] Moreover, antagonists directed against TRP-PLIK2 are useful fordecreasing NF-kB activity, decreasing apoptotic events, and/orincreasing IkBa expression or activity levels.

[0149] In preferred embodiments, agonists directed against TRP-PLIK2 areuseful for treating, diagnosing, and/or ameliorating autoimmunediorders, disorders related to hyper immune activity, hypercongenitalconditions, birth defects, necrotic lesions, wounds, disorders relatedto aberrant signal transduction, immuno compromised conditions, HIVinfection, proliferating disorders, and/or cancers.

[0150] Moreover, agonists directed against TRP-PLIK2 are useful forincreasing NF-kB activity, increasing apoptotic events, and/ordecreasing IkBa expression or activity levels.

[0151] The strong homology to transient receptor potential channels(TRP), combined with the predominate localized expression in the lowergastrointestinal tract, specifically the ileum, the rectum, the colon,the jejunum, and to a lesser extent in the duodenum and stomach,suggests the TRP-PLIK2 polynucleotides and polypeptides may be useful intreating, diagnosing, prognosing, and/or preventing gastrointesinaldiseases and/or disorders, which include, but are not limited to,ulcers, irritable bowel syndrome, inflammatory bowel disease, diarrhea,traveler's diarrhea, drug-related diarrhea polyps, absorption disorders,constipation, diverticulitis, vascular disease of the intestines,intestinal obstruction, intestinal infections, ulcerative colitis,Shigellosis, cholera, Crohn's Disease, amebiasis, enteric fever,Whipple's Disease, peritonitis, intrabdominal abcesses, hereditaryhemochromatosis, gastroenteritis, viral gastroenteritis, food poisoning,mesenteric ischemia, mesenteric infarction, in addition to, metabolicdiseases and/or disorders.

[0152] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosingsusceptibility to the following, non-limiting, gastrointestinalinfections: Salmonella infection, E. coli infection, E.coli 0157:H7infection, Shiga Toxin-producing E. coli infection, Campylobacterinfection (e.g., Campylobacter fetus, Campylobacter upsaliensis,Campylobacter hyointestinalis, Campylobacter lari, Campylobacter jejuni,Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum,Campylobacter rectus, Campylobacter curvus, Campylobacter sputorum,etc.), Heliobacter infection (e.g., Heliobacter cinaedi, Heliobacterfennelliae, etc.)Yersinia enterocolitica infection, Vibrio sp. Infection(e.g., Vibrio mimicus, Vibrio parahaemolyticus, Vibrio fluvialis, Vibriofurnissii, Vibrio hollisae, Vibrio vulnificus, Vibrio alginolyticus,Vibrio metschnikovii, Vibrio damsela, Vibrio cincinnatiensis, etc.)Aeromonas infection (e.g., Aeromonas hydrophila, Aeromonas sobira,Aeromonas caviae, etc.), Plesiomonas shigelliodes infection, Giardiainfection (e.g., Giardia lamblia, etc.), Cryptosporidium infection,Listeria infection, Entamoeba histolytica infection, Rotavirusinfection, Norwalk virus infection, Clostridium difficile infection,Clostriudium perfringens infection, Staphylococcus infection, Bacillusinfection, in addition to any other gastrointestinal disease and/ordisorder implicated by the causative agents listed above or elsewhereherein.

[0153] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in kidney tissuesuggests the TRP-PLIK2 polynucleotides and polypeptides may be useful intreating, diagnosing, prognosing, and/or preventing renal diseasesand/or disorders, which include, but are not limited to: nephritis,renal failure, nephrotic syndrome, urinary tract infection, hematuria,proteinuria, oliguria, polyuria, nocturia, edema, hypertension,electrolyte disorders, sterile pyuria, renal osteodystrophy, largekidneys, renal transport defects, nephrolithiasis, azotemia, anuria,urinary retention slowing of urinary stream, large prostate, flanktenderness, full bladder sensation after voiding, enuresis,dysuria,bacteriuria, kideny stones, glomerulonephritis, vasculitis,hemolytic uremic syndromes, thrombotic thrombocytopenic purpura,malignant hypertension, casts, tubulointerstitial kidney diseases, renaltubular acidosis, pyelonephritis, hydronephritis, nephrotic syndrome,crush syndrome, and/or renal colic, in addition to Wilm's Tumor Disease,and congenital kidney abnormalities such as horseshoe kidney, polycystickidney, and Falconi's syndrome.for example.

[0154] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(1 3):8375-8, (1999)).

[0155] Thus, the TRP-PLIK2 polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in kidney cells and tissues, particularly those specificallyreferenced herein.

[0156] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in bone marrowtissue suggests the TRP-PLIK2 polynucleotides and polypeptides may beuseful in treating, diagnosing, prognosing, and/or preventing immunediseases and/or disorders. Representative uses are described in the“Immune Activity”, “Chemotaxis”, and “Infectious Disease” sectionsbelow, and elsewhere herein. Briefly, the strong expression in immunetissue indicates a role in regulating the proliferation; survival;differentiation; and/or activation of hematopoietic cell lineages,including blood stem cells.

[0157] The TRP-PLIK2 polypeptide may also be useful as a preventativeagent for immunological disorders including arthritis, asthma,immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis,granulomatous disease, inflammatory bowel disease, sepsis, acne,neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cellmediated cytotoxicity; immune reactions to transplanted organs andtissues, such as host-versus-graft and graft-versus-host diseases, orautoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma. The TRP-PLIK2 polypeptide may be useful for modulatingcytokine production, antigen presentation, or other processes, such asfor boosting immune responses, etc.

[0158] Moreover, the protein may represent a factor that influences thedifferentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, astissuemarkers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0159] Significantly, TRP-PLIK2 is believed to represent the first TRPfamily member expressed in bone marrow tissue.

[0160] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in testis tissueemphasizes the potential utility for TRP-PLIK2 polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingtesticular, in addition to reproductive disorders.

[0161] In preferred embodiments, TRP-PLIK2 polynucleotides andpolypeptides including agonists, antagonists, and/or fragments thereof,have uses which include treating, diagnosing, prognosing, and/orpreventing the following, non-limiting, diseases or disorders of thetestis: spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The TRP-PLIK2polynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0162] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for TRP-PLIK2 polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0163] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0164] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I.,Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(l):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Birnbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0165] Thus, the TRP-PLIK2 polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in testis cells and tissues, particularly those specificallyreferenced herein.

[0166] The predominate differential expression of TRP-PLIK2 in prostatetumor relative to normal prostate tissue strongly suggests TRP-PLIK2polynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, have uses which include treating, diagnosing,prognosing, and/or preventing prostate cancers and/or proliferativeconditions.

[0167] Alternatively, the tissue distribution in liver indicates theprotein product of this clone would be useful for the detection andtreatment of liver disorders and cancers. Representative uses aredescribed in the “Hyperproliferative Disorders”, “Infectious Disease”,and “Binding Activity” sections below, and elsewhere herein. Briefly,the protein can be used for the detection, treatment, and/or preventionof hepatoblastoma, jaundice, hepatitis, liver metabolic diseases andconditions that are attributable to the differentiation of hepatocyteprogenitor cells, cirrhosis, hepatic cysts, pyrogenic abscess, amebicabcess, hydatid cyst, cystadenocarcinoma, adenoma, focal nodularhyperplasia, hemangioma, hepatocellulae carcinoma, cholangiocarcinoma,angiosarcoma, and granulomatous liver disease.

[0168] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosing thefollowing, non-limiting, hepatic infections: liver disease caused bysepsis infection, liver disease caused by bacteremia, liver diseasecaused by Pneomococcal pneumonia infection, liver disease caused byToxic shock syndrome, liver disease caused by Listeriosis, liver diseasecaused by Legionnaries' disease, liver disease caused by Brucellosisinfection, liver disease caused by Neisseria gonorrhoeae infection,liver disease caused by Yersinia infection, liver disease caused bySalmonellosis, liver disease caused by Nocardiosis, liver disease causedby Spirochete infection, liver disease caused by Treponema palliduminfection, liver disease caused by Brrelia burgdorferi infection, liverdisease caused by Leptospirosis, liver disease caused by Coxiellaburnetii infection, liver disease caused by Rickettsia richettsiiinfection, liver disease caused by Chlamydia trachomatis infection,liver disease caused by Chiamydia psittaci infection, in addition to anyother hepatic disease and/or disorder implicated by the causative agentslisted above or elsewhere herein.

[0169] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0170] In preferred embodiments, TRP-PLIK2 polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0171] In more preferred embodiments, TRP-PLIK2 polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0172] TRP-PLIK2 polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since TRP-PLIK2 is dominantly expressed in bone marrow, it mayparticularly play an important role in regulating cytosolic Ca²⁺ inimmune system.

[0173] The TRP-PLIK2 gene maps to chromosome 9q21.2-22.1. This region islinked to amyotrophic lateral sclerosis with frontotemporal dementia,early-onset pulverulent cataract, infantile nephronophthisis,hypomagnesemia with secondary hypocalcemia and familial hemophagocyticlymphohistiocytosis. Therefore, agonists and/or antagonists of the novelTRP-PLIK2 can be used to treat diseases including various forms ofneuronal degeneration, neurogenic inflammation, allergy,immunodeficiency/excessive immune activation, visual defects, hearingdisorder, pain, cancer, hypertension and other cardiovascular diseases.In addition, the therapeutics may be useful in the treatment of diseasesassociated with disturbances in Ca²⁺ homeostasis including osteoporosis,hypercalciuric stone disease, and chronic renal failure.

[0174] In addition, TRP-PLIK2 polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ senstive proteins, the activation of Ca++ senstivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0175] The TRP-PLIK2 polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian bone marrow, kidney, testis, liver, small intestine, spinalcord, prostate, uterus, lung, lymph node, stomach, heart, brain, thymus,and pancreas, preferably human. TRP-PLIK2 polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, may be useful in diagnosing, treating, prognosing,and/or preventing immune, hematopoietic, renal, reproductive, hepatic,and/or proliferative diseases or disorders, particularly of the immunesystem.

[0176] In addition, antagonists of the TRP-PLIK2 polynucleotides andpolypeptides may have uses that include diagnosing, treating,prognosing, and/or preventing diseases or disorders related to transientreceptor potential channel activity, which may include immune,hematopoietic, renal, reproductive, hepatic, and/or proliferativediseases or disorders.

[0177] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those from CHAKI, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the TRP-PLIK2polynucleotides, in addition to, other clones of the present invention,may be arrayed on microchips for expression profiling. Depending onwhich polynucleotide probe is used to hybridize to the slides, a changein expression of a specific gene may provide additional insight into thefunction of this gene based upon the conditions being studied. Forexample, an observed increase or decrease in expression levels when thepolynucleotide probe used comes from tissue that has been treated withknown immunoglobulin inhibitors, which include, but are not limited tothe drugs listed herein or otherwise known in the art, might indicate afunction in modulating immunoglobulin function, for example. In the caseof TRP-PLIK2, bone marrow, kidney, testis, liver, small intestine,spinal cord, prostate, uterus, lung, lymph node, stomach, heart, brain,thymus, and/or pancrease, should be used to extract RNA to prepare theprobe.

[0178] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the TRP-PLIK2 gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-G).

[0179] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theTRP-PLIK2, transforming yeast deficient in transient receptor potentialchannel activity with TRP-PLIK2 and assessing their ability to growwould provide convincing evidence the TRP-PLIK2 polypeptide hastransient receptor potential channel activity. Additional assayconditions and methods that may be used in assessing the function of thepolynucletides and polypeptides of the present invention are known inthe art, some of which are disclosed elsewhere herein.

[0180] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0181] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a bone marrow,kidney, testis, liver, small intestine, spinal cord, prostate, uterus,lung, lymph node, stomach, heart, brain, thymus, and/orpancrease-specific promoter), or it can be expressed at a specified timeof development using an inducible and/or a developmentally regulatedpromoter.

[0182] In the case of TRP-PLIK2 transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (immune, hematopoietic, renal, reproductive,hepatic, or proliferative disorders, etc.) may lead to understanding thefunction of the gene. Therefore, the application of antisense and/orsense methodology to the creation of transgenic mice or rats to refinethe biological function of the polypeptide is encompassed by the presentinvention.

[0183] In preferred embodiments, the following N-terminal TRP-PLIK2deletion polypeptides are encompassed by the present invention:M1-L2017, 12-L2017, 13-L2017, L4-L2017, S5-L2017, K6-L2017, S7-L2017,Q8-L2017, K9-L2017, S10-L2017, W11-L2017, 112-L2017, K13-L2017,G14-L2017, V15-L2017, F16-L2017, D17-L2017, K18-L2017, R19-L2017,E20-L2017, C21-L2017, S22-L2017, T23-L2017, 124-L2017, 125-L2017,P26-L2017, S27-L2017, S28-L2017, K29-L2017, N30-L2017, P31-L2017,H32-L2017, R33-L2017, C34-L2017, T35-L2017, P36-L2017, V37-L2017,C38-L2017, Q39-L2017, V40-L2017, C41-L2017, Q42-L2017, N43-L2017,L44-L2017, 145-L2017, R46-L2017, C47-L2017, Y48-L2017, C49-L2017,G50-L2017, R51-L2017, L52-L2017, 153-L2017, G54-L2017, D55-L2017,H56-L2017, A57-L2017, G58-L2017, 159-L2017, D60-L2017, Y61-L2017,S62-L2017, W63-L2017, T64-L2017, 165-L2017, S66-L2017, A67-L2017,A68-L2017, K69-L2017, G70-L2017, K71-L2017, E72-L2017, S73-L2017,E74-L2017, Q75-L2017, W76-L2017, S77-L2017, V78-L2017, E79-L2017,K80-L2017, H81-L2017, T82-L2017, T83-L2017, K84-L2017, S85-L2017,P86-L2017, T87-L2017, D88-L2017, T89-L2017, F90-L2017, G91-L2017,T92-L2017, 193-L2017, N94-L2017, F95-L2017, Q96-L2017, D97-L2017,G98-L2017, E99-L2017, H100-L2017, T10-L2017, H102-L2017, H103-L2017,A104-L2017, K105-L2017, Y106-L2017, 1107-L2017, R108-L2017, T109-L2017,S110-L2017, Y11 -L2017, D112-L2017, T113-L2017, K114-L2017, L115-L2017,D116-L2017, H117-L2017, L118-L2017, L119-L2017, H120-L2017, L121-L2017,M122-L2017, L123-L2017, K124-L2017, E125-L2017, W126-L2017, K127-L2017,M128-L2017, E129-L2017, L130-L2017, P131-L2017, K132-L2017, L133-L2017,V134-L2017, 1135-L2017, S136-L2017, V137-L2017, H138-L2017, G139-L2017,G140-L2017, 1141-L2017, Q142-L2017, N143-L2017, F144-L2017, T145-L2017,M146-L2017, P147-L2017, S148-L2017, K149-L2017, F150-L2017, K151-L2017,E152-L2017, 1153-L2017, F154-L2017, S155-L2017, Q156-L2017, G157-L2017,L158-L2017, V159-L2017, K160-L2017, A161-L2017, A162-L2017, E163-L2017,T164-L2017, T165-L2017, G166-L2017, A167-L2017, W168-L2017, 1169-L2017,1170-L2017, T171-L2017, E172-L2017, G173-L2017, 1174-L2017, N175-L2017,T176-L2017, G177-L2017, V178-L2017, S179-L2017, K180-L2017, H181-L2017,V182-L2017, G183-L2017, D184-L2017, A185-L2017, L186-L2017, K187-L2017,S188-L2017, H189-L2017, S190-L2017, S191-L2017, H192-L2017, S193-L2017,L194-L2017, R195-L2017, K196-L2017, 1197-L2017, W198-L2017, T199-L2017,V200-L2017, G201-L2017, 1202-L2017, P203-L2017, P204-L2017, W205-L2017,G206-L2017, V207-L2017, 1208-L2017, E209-L2017, N210-L2017, Q211-L2017,R212-L2017, D213-L2017, L214-L2017, 1215-L2017, G216-L2017, K217-L2017,D218-L2017, V219-L2017, V220-L2017, C221-L2017, L222-L2017, Y223-L2017,Q224-L2017, T225-L2017, L226-L2017, D227-L2017, N228-L2017, P229-L2017,L230-L2017, S231-L2017, K232-L2017, L233-L2017, T234-L2017, T235-L2017,L236-L2017, N237-L2017, S238-L2017, M239-L2017, H240-L2017, S241 -L2017,H242-L2017, F243-L2017, I244-L2017, L245-L2017, S246-L2017, D247-L2017,D248-L2017, G249-L2017, T250-L2017, V251-L2017, G252-L2017, K253-L2017,Y254-L2017, G255-L2017, N256-L2017, E257-L2017, M258-L2017, K259-L2017,L260-L2017, R261-L2017, R262-L2017, N263-L2017, L264-L2017, E265-L2017,K266-L2017, Y267-L2017, L268-L2017, S269-L2017, L270-L2017, Q271-L2017,K272-L2017, 1273-L2017, H274-L2017, C275-L2017, R276-L2017, S277-L2017,R278-L2017, Q279-L2017, G280-L2017, V281-L2017, P282-L2017, V283-L2017,V284-L2017, G285-L2017, L286-L2017, V287-L2017, V288-L2017, E289-L2017,G290-L2017, G291-L2017, P292-L2017, N293-L2017, V294-L2017, 1295-L2017,L296-L2017, S297-L2017, V298-L2017, W299-L2017, E300-L2017, T301-L2017,V302-L2017, K303-L2017, D304-L2017, K305-L2017, D306-L2017, P307-L2017,V308-L2017, V309-L2017, V310-L2017, C311-L2017, E312-L2017, G313-L2017,T314-L2017, G315-L2017, R316-L2017, A317-L2017, A318-L2017, D319-L2017,L320-L2017, L321-L2017, A322-L2017, F323-L2017, T324-L2017, H325-L2017,K326-L2017, H327-L2017, L328-L2017, A329-L2017, D330-L2017, E331-L2017,G332-L2017, M333-L2017, L334-L2017, R335-L2017, P336-L2017, Q337-L2017,V338-L2017, K339-L2017, E340-L2017, E341-L2017, 1342-L2017, 1343-L2017,C344-L2017, M345-L2017, 1346-L2017, Q347-L2017, N348-L2017, T349-L2017,F350-L2017, N351-L2017, F352-L2017, S353-L2017, L354-L2017, K355-L2017,Q356-L2017, S357-L2017, K358-L2017, H359-L2017, L360-L2017, F361-L2017,Q362-L2017, 1363-L2017, L364-L2017, M365-L2017, E366-L2017, C367-L2017,M368-L2017, V369-L2017, H370-L2017, R371-L2017, D372-L2017, C373-L2017,1374-L2017, T375-L2017, 1376-L2017, F377-L2017, D378-L2017, A379-L2017,D380-L2017, S381-L2017, E382-L2017, E383-L2017, Q384-L2017, Q385-L2017,D386-L2017, L387-L2017, D388-L2017, L389-L2017, A390-L2017, 1391-L2017,L392-L2017, T393-L2017, A394-L2017, L395-L2017, L396-L2017, K397-L2017,G398-L2017, T399-L2017, N400-L2017, L401-L2017, S402-L2017, A403-L2017,S404-L2017, E405-L2017, Q406-L2017, L407-L2017, N408-L2017, L409-L2017,A410-L2017, M411-L2017, A412-L2017, W413-L2017, D414-L2017, R415-L2017,V416-L2017, D417-L2017, 1418-L2017, A419-L2017, K420-L2017, K421-L2017,H422-L2017, 1423-L2017, L424-L2017, 1425-L2017, Y426-L2017, E427-L2017,Q428-L2017, H429-L2017, W430-L2017, K431-L2017, P432-L2017, D433-L2017,A434-L2017, L435-L2017, E436-L2017, Q437-L2017, A438-L2017, M439-L2017,S440-L2017, D441-L2017, A442-L2017, IA43-L2017, V444-L2017, M445-L2017,D446-L2017, R447-L2017, V448-L2017, D449-L2017, F450-L2017, V451-L2017,K452-L2017, L453-L2017, L454-L2017, 1455-L2017, E456-L2017, Y457-L2017,G458-L2017, V459-L2017, N460-L2017, L461-L2017, H462-L2017, R463-L2017,F464-L2017, L465-L2017, T466-L2017, 1467-L2017, P468-L2017, R469-L2017,L470-L2017, E471-L2017, E472-L2017, L473-L2017, Y474-L2017, N475-L2017,T476-L2017, K477-L2017, Q478-L2017, G479-L2017, P480-L2017, T481-L2017,N482-L2017, T483-L2017, L484-L2017, L485-L2017, H486-L2017, H487-L2017,L488-L2017, V489 L2017, V492-L2017, K493-L2017, Q494-L2017, H495-L2017,T496-L2017, L497-L2017, L498-L2017, S499-L2017, G500-L2017, Y501-L2017,R502-L2017, 1503-L2017, T504-L2017, L505-L2017, 1506-L2017, D507-L2017,1508-L2017, G509-L2017, L510-L2017, V511-L2017, V512-L2017, E513-LL2017, 1516-L2017, G517-L2017, R518-L2017, A519-L2017, Y520-L2017,R521-L2017, S522-L2017, N523-L2017, Y524-L2017, T525-L2017, R526-L2017,K527-L2017, H528-L2017, F529-L2017, R530-L2017, A531-L2017, L532-L2017,Y533-L2017, N534-L2017, N535-L2017, L536-L2017, Y537-L2017, R538-L2017,K539-L2017, Y540-L2017, K541-L2017, H542-L2017, Q543-L2017, R544-L2017,H545-L2017, S546-L2017, S547-L2017, G548-L2017, N549-L2017, R550-L2017,N551-L2017, E552-L2017, S553-L2017, A554-L2017, E555-L2017, S556-L2017,T557-L2017, L558-L2017, H559-L2017, S560-L2017, Q561-L2017, F562-L2017,1563-L2017, R564-L2017, T565-L2017, A566-L2017, Q567-L2017, P568-L2017,Y569-L2017, K570-L2017, F571-L2017, K572-L2017, E573-L2017, K574-L2017,S575-L2017, 1576-L2017, V577-L2017, L578-L2017, H579-L2017, K580-L2017,S581-L2017, R582-L2017, K583-L2017, K584-L2017, S585-L2017, K586-L2017,E587-L2017, Q588-L2017, N589-L2017, V590-L2017, S591-L2017, D592-L2017,D593-L2017, P594-L2017, E595-L2017, S596-L2017, T597-L2017, G598-L2017,F599-L2017, L600-L2017, Y601-L2017, P602-L2017, Y603-L2017, N604-L2017,D605-L2017, L606-L2017, L607-L2017, V608-L2017, W609-L2017, A610-L2017,V611-L2017, L612-L2017, M613-L2017, K614-L2017, R615-L2017, Q616-L2017,K617-L2017, M618-L2017, A619-L2017, M620-L2017, F621-L2017, F622-L2017,W623-L2017, Q624-L2017, H625-L2017, G626-L2017, E627-L2017, E628-L2017,A629-L2017, T630-L2017, V631-L2017, K632-L2017, A633-L2017, V634-L2017,1635-L2017, A636-L2017, C637-L2017, 1638-L2017, L639-L2017, Y640-L2017,R641-L2017, A642-L2017, M643-L2017, A644-L2017, H645-L2017, E646-L2017,A647-L2017, K648-L2017, E649-L2017, S650-L2017, H651-L2017, M652-L2017,V653-L2017, D654-L2017, D655-L2017, A656-L2017, S657-L2017, E658-L2017,E659-L2017, L660-L2017, K661-L2017, N662-L2017, Y663-L2017, S664-L2017,K665-L2017, Q666-L2017, F667-L2017, G668-L2017, Q669-L2017, L670-L2017,A671-L2017, L672-L2017, D673-L2017, L674-L2017, L675-L2017, E676-L2017,K677-L2017, A678-L2017, F679-L2017, K680-L2017, Q681-L2017, N682-L2017,E683-L2017, R684-L2017, M685-L2017, A686-L2017, M687-L2017, T688-L2017,L689-L2017, L690-L2017, T691-L2017, Y692-L2017, E693-L2017, L694-L2017,R695-L2017, N696-L2017, W697-L2017, S698-L2017, N699-L2017, S700-L2017,T701-L2017, C702-L2017, L703-L2017, K704-L2017, L705-L2017, A706-L2017,V707-L2017, S708-L2017, G709-L2017, G710-L2017, L711-L2017, R712-L2017,P713-L2017, F714-L2017, V715-L2017, S716-L2017, H717-L2017, T718-L2017,C719-L2017, T720-L2017, Q721-L2017, M722-L2017, L723-L2017, L724-L2017,T725-L2017, D726-L2017, M727-L2017, W728-L2017, M729-L2017, G730-L2017,R731-L2017, L732-L2017, K733-L2017, M734-L2017, R735-L2017, K736-L2017,N737-L2017, S738-L2017, W739-L2017, L740-L2017, K741-L2017, 1742-L2017,1743-L2017, 1744-L2017, S745-L2017, 1746-L2017, 1747-L2017, L748-L2017,P749-L2017, P750-L2017, T751-L2017, 1752-L2017, L753-L2017, T754-L2017,L755-L2017, E756-L2017, F757-L2017, K758-L2017, S759-L2017, K760-L2017,A761-L2017, E762-L2017, M763-L2017, S764-L2017, H765-L2017, V766-L2017,P767-L2017, Q768-L2017, S769-L2017, Q770-L2017, D771-L2017, F772-L2017,Q773-L2017, F774-L2017, M775-L2017, W776-L2017, Y777-L2017, Y778-L2017,S779-L2017, D780-L2017, Q781-L2017, N782-L2017, A783-L2017, S784-L2017,S785-L2017, S786-L2017, K787-L2017, E788-L2017, S789-L2017, A790-L2017,S791-L2017, V792-L2017, K793-L2017, E794-L2017, Y795-L2017, D796-L2017,L797-L2017, E798-L2017, R799-L2017, G800-L2017, H801-L2017, D802-L2017,E803-L2017, K804-L2017, L805-L2017, D806-L2017, E807-L2017, N808-L2017,Q809-L2017, H810-L2017, F811-L2017, G812-L2017, L813-L2017, E814-L2017,S815-L2017, G816-L2017, H817-L2017, Q818-L2017, H819-L2017, L820-L2017,P821-L2017, W822-L2017, T823-L2017, R824-L2017, K825-L2017, V826-L2017,Y827-L2017, E828-L2017, F829-L2017, Y830-L2017, S831-L2017, A832-L2017,P833-L2017, 1834-L2017, V835-L2017, K836-L2017, F837-L2017, W838-L2017,F839-L2017, Y840-L2017, T841-L2017, M842-L2017, A843-L2017, Y844-L2017,L845-L2017, A846-L2017, F847-L2017, L848-L2017, M849-L2017, L850-L2017,F851-L2017, T852-L2017, Y853-L2017, T854-L2017, V855-L2017, L856-L2017,V857-L2017, E858-L2017, M859-L2017, Q860-L2017, P861-L2017, Q862-L2017,P863-L2017, S864-L2017, V865-L2017, Q866-L2017, E867-L2017, W868-L2017,L869-L2017, V870-L2017, S871-L2017, 1872-L2017, Y873-L2017, 1874-L2017,F875-L2017, T876-L2017, N877-L2017, A878-L2017, 1879-L2017, E880-L2017,V881-L2017, V882-L2017, R883-L2017, E884-L2017, 1885-L2017, C886-L2017,1887-L2017, S888-L2017, E889-L2017, P890-L2017, G891-L2017, K892-L2017,F893-L2017, T894-L2017, Q895-L2017, K896-L2017, V897-L2017, K898-L2017,V899-L2017, W900-L2017, 1901-L2017, S902-L2017, E903-L2017, Y904-L2017,W905-L2017, N906-L2017, L907-L2017, T908-L2017, E909-L2017, T910-L2017,V911-L2017, A912-L2017, 1913-L2017, G914-L2017, L915-L2017, F916-L2017,S917-L2017, A918-L2017, G919-L2017, F920-L2017, V921-L2017, L922-L2017,R923-L2017, W924-L2017, G925-L2017, D926-L2017, P927-L2017, P928-L2017,F929-L2017, H930-L2017, T931-L2017, A932-L2017, G933-L2017, R934-L2017,L935-L2017, 1936-L2017, Y937-L2017, C938-L2017, 1939-L2017, D940-L2017,1941-L2017, 1942-L2017, F943-L2017, W944-L2017, F945-L2017, S946-L2017,R947-L2017, L948-L2017, L949-L2017, D950-L2017, F951-L2017, F952-L2017,A953-L2017, V954-L2017, N955-L2017, Q956-L2017, H957-L2017, A958-L2017,G959-L2017, P960-L2017, Y961-L2017, V962-L2017, T963-L2017, M964-L2017,1965-L2017, A966-L2017, K967-L2017, M968-L2017, T969-L2017, A970-L2017,N971-L2017, M972-L2017, F973-L2017, Y974-L2017, 1975-L2017, V976-L2017,1977-L2017, 1978-L2017, M979-L2017, A980-L2017, 1981-L2017, V982-L2017,L983-L2017, L984-L2017, S985-L2017, F986-L2017, G987-L2017, V988-L2017,A989-L2017, R990-L2017, K991-L2017, A992-L2017, 1993-L2017, L994-L2017,S995-L2017, P996-L2017, K997-L2017, E998-L2017, P999-L2017, P1000-L2017,S1001-L2017, W1002-L2017, S1003-L2017, L1004-L2017, A1005-L2017,R1006-L2017, D1007-L2017, 11008-L2017, V1009-L2017, F1O1O-L2017,EIO11-L2017, P1012-L2017, Y1013-L2017, W1014-L2017, M1015-L2017,11016-L2017, Y1017-L2017, G1018-L2017, E1019-L2017, V1020-L2017,Y1021-L2017, A1022-L2017, G1023-L2017, E1024-L2017, 11025-L2017,D1026-L2017, V1027-L2017, C1028-L2017, S1029-L2017, Sl030-L2017,Q1031-L2017, P1032-L2017, S1033-L2017, C1034-L2017, P1035-L2017,P1036-L2017, G1037-L2017, S1038-L2017, F1039-L2017, L1040-L2017,T1041-L2017, P1042-L2017, F1043-L2017, L1044-L2017, Q1045-L2017,A1046-L2017, V1047-L2017, Y1048-L2017, L1049-L2017, F1050-L2017,V1051-L2017, Q1052-L2017, Y1053-L2017, 11054-L2017, 11055-L2017,M1056-L2017, V1057-L2017, N1058-L2017, L1059-L2017, L1060-L2017,11061-L2017, A1062-L2017, F1063-L2017, F1064-L2017, N1065-L2017,N1066-L2017, V1067-L2017, Y1068-L2017, L1069-L2017, D1070-L2017,M1071-L2017, E1072-L2017, S1073-L2017, 11074-L2017, S1075-L2017,N1076-L2017, N1077-L2017, L1078-L2017, W1079-L2017, K1080-L2017,Y1081-L2017, N1082-L2017, R1083-L2017, Y1084-L2017, R1085-L2017,Y1086-L2017, 11087-L2017, M1088-L2017, T1089-L2017, Y1090-L2017,H1091-L2017, E1092-L2017, K1093-L2017, P1094-L2017, W1095-L2017,L1096-L2017, P1097-L2017, P1098-L2017, P1099-L2017, L1100-L2017,11O1-L2017, L1102-L2017, L1103-L2017, S1104-L2017, H1105-L2017,V1106-L2017, G1107-L2017, L1108-L2017, L1109-L2017, L1110-L2017,R1111-L2017, R1112-L2017, L1113-L2017, C1114-L2017, C1115-L2017,H1116-L2017, R1117-L2017, A1118-L2017, P1119-L2017, H1120-L2017,D1121-L2017, Q1122-L2017, E1123-L2017, E1124-L2017, G1125-L2017,D1126-L2017, V1127-L2017, G1128-L2017, L1129-L2017, K1130-L2017,L1131-L2017, Y1132-L2017, L1133-L2017, S1134-L2017, K1135-L2017,E1136-L2017, D1137-L2017, L1138-L2017, K1139-L2017, K1140-L2017,L1141-L2017, H1142-L2017, D1143-L2017, F1144-L2017, E1145-L2017,E1146-L2017, Q1147-L2017, C1148-L2017, V1149-L2017, E1150-L2017,K1151-L2017, Y1152-L2017, F1153-L2017, H1154-L2017, E1155-L2017,K1156-L2017, M1157-L2017, E1158-L2017, D1159-L2017, V1160-L2017,N1161-L2017, C1162-L2017, S1163-L2017, C1164-L2017, E1165-L2017,E1166-L2017, R1167-L2017, 11168-L2017, R1169-L2017, V1170-L2017,T1171-L2017, S1172-L2017, E1173-L2017, R1174-L2017, V1175-L2017,T1176-L2017, E1177-L2017, M1178-L2017, Y1179-L2017, F1180-L2017,Q1181-L2017, L1182-L2017, K1183-L2017, E1184-L2017, M1185-L2017,N1186-L2017, El 187-L2017, KI 188-L2017, V1189-L2017, S1190-L2017,F1191-L2017, 11192-L2017, K1193-L2017, D1194-L2017, S1195-L2017,L1196-L2017, L1197-L2017, S1198-L2017, L1199-L2017, D1200-L2017,S1201-L2017, Q1202-L2017, V1203-L2017, G1204-L2017, H1205-L2017,L1206-L2017, Q1207-L2017, D1208-L2017, L1209-L2017, S1210-L2017,A1211-L2017, L1212-L2017, T1213-L2017, V1214-L2017, D1215-L2017,T1216-L2017, L1217-L2017, K1218-L2017, V1219-L2017, L1220-L2017,S1221-L2017, A1222-L2017, V1223-L2017, D1224-L2017, T1225-L2017,L1226-L2017, Q1227-L2017, E1228-L2017, D1229-L2017, E1230-L2017,A1231-L2017, L1232-L2017, L1233-L2017, A1234-L2017, K1235-L2017,R1236-L2017, K1237-L2017, H1238-L2017, S1239-L2017, T1240-L2017,C1241-L2017, K1242-L2017, K1243-L2017, L1244-L2017, P1245-L2017,H1246-L2017, S1247-L2017, W1248-L2017, S1249-L2017, N1250-L2017,V1251-L2017, 11252-L2017, C1253-L2017, A1254-L2017, E1255-L2017,V1256-L2017, L1257-L2017, G1258-L2017, S1259-L2017, M1260-L2017,E1261-L2017, 11262-L2017, A1263-L2017, G1264-L2017, E1265-L2017,K1266-L2017, K1267-L2017, Y1268-L2017, Q1269-L2017, Y1270-L2017,Y1271-L2017, S1272-L2017, M1273-L2017, P1274-L2017, S1275-L2017,S1276-L2017, L1277-L2017, L1278-L2017, R1279-L2017, S1280-L2017,L1281-L2017, A1282-L2017, G1283-L2017, G1284-L2017, R1285-L2017,H1286-L2017, P1287-L2017, P1288-L2017, R1289-L2017, V1290-L2017,Q1291-L2017, R1292-L2017, G1293-L2017, A1294-L2017, L1295-L2017,L1296-L2017, E1297-L2017, L1298-L2017, T1299-L2017, N1300-L2017,S1301-L2017, K1302-L2017, R1303-L2017, E1304-L2017, A1305-L2017,T1306-L2017, N1307-L2017, V1308-L2017, R1309-L2017, N1310-L2017,D1311-L2017, Q1312-L2017, E1313-L2017, R1314-L2017, Q1315-L2017,E1316-L2017, T1317-L2017, Q1318-L2017, S1319-L2017, S1320-L2017,I1321-L2017, V1322-L2017, V1323-L2017, S1324-L2017, G1325-L2017,V1326-L2017, S1327-L2017, P1328-L2017, N1329-L2017, R1330-L2017,Q1331-L2017, A1332-L2017, H1333-L2017, S1334-L2017, K1335-L2017,Y1336-L2017, G1337-L2017, Q1338-L2017, F1339-L2017, L1340-L2017,L1341-L2017, V1342-L2017, P1343-L2017, S1344-L2017, N1345-L2017,L1346-L2017, K1347-L2017, R1348-L2017, V1349-L2017, P1350-L2017,F1351-L2017, S1352-L2017, A1353-L2017, E1354-L2017, T1355-L2017,V1356-L2017, L1357-L2017, P1358-L2017, L1359-L2017, S1360-L2017,R1361-L2017, P1362-L2017, S1363-L2017, V1364-L2017, P1365-L2017,D1366-L2017, V1367-L2017, L1368-L2017, A1369-L2017, T1370-L2017,E1371-L2017, Q1372-L2017, D1373-L2017, 11374-L2017, Q1375-L2017,T1376-L2017, E1377-L2017, V1378-L2017, L1379-L2017, V1380-L2017,H1381-L2017, L1382-L2017, T1383-L2017, G1384-L2017, Q1385-L2017,T1386-L2017, P1387-L2017, V1388-L2017, V1389-L2017, S1390-L2017,D1391-L2017, W1392-L2017, A1393-L2017, S1394-L2017, V1395-L2017,D1396-L2017, E1397-L2017, P1398-L2017, K1399-L2017, E1400-L2017,K1401-L2017, H1402-L2017, E1403-L2017, P1404-L2017, 11405-L2017,A1406-L2017, H1407-L2017, L1408-L2017, L1409-L2017, D1410-L2017,G1411-L2017, Q1412-L2017, D1413-L2017, K1414-L2017, A1415-L2017,E1416-L2017, Q1417-L2017, V1418-L2017, L1419-L2017, P1420-L2017,T1421-L2017, L1422-L2017, S1423-L2017, C1424-L2017, T1425-L2017,P1426-L2017, E1427-L2017, P1428-L2017, M1429-L2017, T1430-L2017,M1431-L2017, S1432-L2017, S1433-L2017, P1434-L2017, L1435-L2017,S1436-L2017, Q1437-L2017, A1438-L2017, K1439-L2017, 11440-L2017,M1441-L2017, Q1442-L2017, T1443-L2017, G1444-L2017, G1445-L2017,G1446-L2017, Y1447-L2017, V1448-L2017, N1449-L2017, W1450-L2017,A1451-L2017, F1452-L2017, S1453-L2017, E1454-L2017, G1455-L2017,D1456-L2017, E1457-L2017, T1458-L2017, G1459-L2017, V1460-L2017,F1461-L2017, S1462-L2017, 11463-L2017, K1464-L2017, K1465-L2017,K1466-L2017, W1467-L2017, Q1468-L2017, T1469-L2017, C1470-L2017,L1471-L2017, P1472-L2017, S1473-L2017, T1474-L2017, C1475-L2017,D1476-L2017, S1477-L2017, D1478-L2017, S1479-L2017, S1480-L2017,R1481-L2017, S1482-L2017, E1483-L2017, Q1484-L2017, H1485-L2017,Q1486-L2017, K1487-L2017, Q1488-L2017, A1489-L2017, Q1490-L2017,D1491-L2017, S1492-L2017, S1493-L2017, L1494-L2017, S1495-L2017,D1496-L2017, N1497-L2017, S1498-L2017, T1499-L2017, R1500-L2017,S1501-L2017, A1502-L2017, Q1503-L2017, S1504-L2017, S1505-L2017,E1506-L2017, C1507-L2017, S1508-L2017, E1509-L2017, V1510-L2017,G1511-L2017, P1512-L2017, W1513-L2017, L1514-L2017, Q1515-L2017,P1516-L2017, N1517-L2017, T1518-L2017, S1519-L2017, F1520-L2017,W1521-L2017, I1522-L2017, N1523-L2017, P1524-L2017, L1525-L2017,R1526-L2017, R1527-L2017, Y1528-L2017, R1529-L2017, P1530-L2017,F1531-L2017, A1532-L2017, R1533-L2017, S1534-L2017, H1535-L2017,S1536-L2017, F1537-L2017, R1538-L2017, F1539-L2017, H1540-L2017,K1541-L2017, E1542-L2017, E1543-L2017, K1544-L2017, L1545-L2017,M1546-L2017, K1547-L2017, 11548-L2017, C1549-L2017, K1550-L2017,11551-L2017, K1552-L2017, N1553-L2017, L1554-L2017, S1555-L2017,G1556-L2017, S1557-L2017, S1558-L2017, E1559-L2017, 11560-L2017,G1561-L2017, Q1562-L2017, G1563-L2017, A1564-L2017, W1565-L2017,V1566-L2017, K1567-L2017, A1568-L2017, K1569-L2017, M1570-L2017,L1571-L2017, T1572-L2017, K1573-L2017, D1574-L2017, R1575-L2017,R1576-L2017, L1577-L2017, S1578-L2017, K1579-L2017, K1580-L2017,K1581-L2017, K1582-L2017, N1583-L2017, T1584-L2017, Q1585-L2017,G1586-L2017, L1587-L2017, Q1588-L2017, V1589-L2017, P1590-L2017,11591-L2017, 11592-L2017, T1593-L2017, V1594-L2017, N1595-L2017,A1596-L2017, C1597-L2017, S1598-L2017, Q1599-L2017, S1600-L2017,D1601-L2017, Q1602-L2017, L1603-L2017, N1604-L2017, P1605-L2017,E1606-L2017, P1607-L2017, G1608-L2017, E1609-L2017, N1610-L2017,S1611-L2017, 11612-L2017, S1613-L2017, E1614-L2017, E1615-L2017,E1616-L2017, Y1617-L2017, S1618-L2017, K1619-L2017, N1620-L2017,W1621-L2017, F1622-L2017, T1623-L2017, V1624-L2017, S1625-L2017,K1626-L2017, F1627-L2017, S1628-L2017, H1629-L2017, T1630-L2017,G1631-L2017, V1632-L2017, E1633-L2017, P1634-L2017, Y1635-L2017,11636-L2017, H1637-L2017, Q1638-L2017, K1639-L2017, M1640-L2017,K1641-L2017, T1642-L2017, K1643-L2017, E1644-L2017, 11645-L2017,G1646-L2017, Q1647-L2017, C1648-L2017, A1649-L2017, 11650-L2017,Q1651-L2017, I1652-L2017, S1653-L2017, D1654-L2017, Y1655-L2017,L1656-L2017, K1657-L2017, Q1658-L2017, S1659-L2017, Q1660-L2017,E1661-L2017, D1662-L2017, L1663-L2017, S1664-L2017, K1665-L2017,N1666-L2017, S1667-L2017, L1668-L2017, W1669-L2017, N1670-L2017,S1671-L2017, R1672-L2017, S1673-L2017, T1674-L2017, N1675-L2017,L1676-L2017, N1677-L2017, R1678-L2017, N1679-L2017, S1680-L2017,L1681-L2017, L1682-L2017, K1683-L2017, S1684-L2017, S1685-L2017,11686-L2017, G1687-L2017, V1688-L2017, D1689-L2017, K1690-L2017,11691-L2017, S1692-L2017, A1693-L2017, S1694-L2017, L1695-L2017,K1696-L2017, S1697-L2017, P1698-L2017, Q1699-L2017, E1700-L2017,P1701-L2017, H1702-L2017, H1703-L2017, H1704-L2017, Y1705-L2017,S1706-L2017, A1707-L2017, 11708-L2017, E1709-L2017, R1710-L2017,N1711-L2017, N1712-L2017, L1713-L2017, M1714-L2017, R1715-L2017,L1716-L2017, S1717-L2017, Q1718-L2017, T1719-L2017, 11720-L2017,P1721-L2017, F1722-L2017, T1723-L2017, P1724-L2017, V1725-L2017,Q1726-L2017, L1727-L2017, F1728-L2017, A1729-L2017, G1730-L2017,E1731-L2017, E1732-L2017, I1733-L2017, T1734-L2017, V1735-L2017,Y1736-L2017, R1737-L2017, L1738-L2017, E1739-L2017, E1740-L2017,S1741-L2017, S1742-L2017, P1743-L2017, L1744-L2017, N1745-L2017,L1746-L2017, D1747-L2017, K1748-L2017, S1749-L2017, M1750-L2017,S1751-L2017, S1752-L2017, W1753-L2017, S1754-L2017, Q1755-L2017,R1756-L2017, G1757-L2017, R1758-L2017, A1759-L2017, A1760-L2017,M1761-L2017, 11762-L2017, Q1763-L2017, V1764-L2017, L1765-L2017,S1766-L2017, R1767-L2017, E1768-L2017, E1769-L2017, M1770-L2017,D1771-L2017, G1772-L2017, G1773-L2017, L1774-L2017, R1775-L2017,K1776-L2017, A1777-L2017, M1778-L2017, R1779-L2017, V1780-L2017,V1781-L2017, S1782-L2017, T1783-L2017, W1784-L2017, S1785-L2017,E1786-L2017, D1787-L2017, D1788-L2017, 11789-L2017, L1790-L2017,K1791-L2017, P1792-L2017, G1793-L2017, Q1794-L2017, V1795-L2017,F1796-L2017, 11797-L2017, V1798-L2017, K1799-L2017, S1800-L2017,F1801-L2017, L1802-L2017, P1803-L2017, E1804-L2017, V1805-L2017,V1806-L2017, R1807-L2017, T1808-L2017, W1809-L2017, H1810-L2017,K1811-L2017, 11812-L2017, F1813-L2017, Q1814-L2017, E1815-L2017,S1816-L2017, T1817-L2017, V1818-L2017, L1819-L2017, H1820-L2017,L1821-L2017, C1822-L2017, L1823-L2017, R1824-L2017, E1825-L2017,11826-L2017, Q1827-L2017, Q1828-L2017, Q1829-L2017, R1830-L2017,A1831-L2017, A1832-L2017, Q1833-L2017, K1834-L2017, L1835-L2017,11836-L2017, Y1837-L2017, T1838-L2017, F1839-L2017, N1840-L2017,Q1841-L2017, V1842-L2017, K1843-L2017, P1844-L2017, Q1845-L2017,T1846-L2017, 11847-L2017, P1848-L2017, Y1849-L2017, T1850-L2017,P1851-L2017, R1852-L2017, F1853-L2017, L1854-L2017, E1855-L2017,V1856-L2017, F1857-L2017, L1858-L2017, I1859-L2017, Y1860-L2017,C1861-L2017, H1862-L2017, S1863-L2017, A1864-L2017, N1865-L2017,Q1866-L2017, W1867-L2017, L1868-L2017, T1869-L2017, 11870-L2017,E1871-L2017, K1872-L2017, Y1873-L2017, M1874-L2017, T1875-L2017,G1876-L2017, E1877-L2017, F1878-L2017, R1879-L2017, K1880-L2017,Y1881-L2017, N1882-L2017, N1883-L2017, N1884-L2017, N1885-L2017,G1886-L2017, D1887-L2017, E1888-L2017, I1889-L2017, T1890-L2017,P1891-L2017, T1892-L2017, N1893-L2017, T1894-L2017, L1895-L2017,E1896-L2017, E1897-L2017, L1898-L2017, M1899-L2017, L1900-L2017,A1901-L2017, F1902-L2017, S1903-L2017, H1904-L2017, W1905-L2017,T1906-L2017, Y1907-L2017, E1908-L2017, Y1909-L2017, T1910-L2017,R1911-L2017, G1912-L2017, E1913-L2017, L1914-L2017, L1915-L2017,V1916-L2017, L1917-L2017, D1918-L2017, L1919-L2017, Q1920-L2017,G1921-L2017, V1922-L2017, G1923-L2017, E1924-L2017, N1925-L2017,L1926-L2017, T1927-L2017, D1928-L2017, P1929-L2017, S1930-L2017,V1931-L2017, 11932-L2017, K1933-L2017, P1934-L2017, E1935-L2017,V1936-L2017, K1937-L2017, Q1938-L2017, S1939-L2017, R1940-L2017,G1941-L2017, M1942-L2017, V1943-L2017, F1944-L2017, G1945-L2017,P1946-L2017, A1947-L2017, N1948-L2017, L1949-L2017, G1950-L2017,E1951-L2017, D1952-L2017, A1953-L2017, 11954-L2017, R1955-L2017,N1956-L2017, F1957-L2017, 11958-L2017, A1959-L2017, K1960-L2017,H1961-L2017, H1962-L2017, C1963-L2017, N1964-L2017, S1965-L2017,C1966-L2017, C1967-L2017, R1968-L2017, K1969-L2017, L1970-L2017,K1971-L2017, L1972-L2017, P1973-L2017, D1974-L2017, L1975-L2017,K1976-L2017, R1977-L2017, N1978-L2017, D1979-L2017, Y1980-L2017,S1981-L2017, P1982-L2017, E1983-L2017, R1984-L2017, 11985-L2017,N1986-L2017, S1987-L2017, T1988-L2017, F1989-L2017, G1990-L2017,L1991-L2017, E1992-L2017, 11993-L2017, K1994-L2017, 11995-L2017,E1996-L2017, S1997-L2017, A1998-L2017, E1999-L2017, E2000-L2017,P2001-L2017, P2002-L2017, A2003-L2017, R2004-L2017, E2005-L2017,T2006-L2017, G2007-L2017, R2008-L2017, N2009-L2017, S2010-L2017, and/orP2011-L2017 of SEQ ID NO:2. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal TRP-PLIK2 deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0184] In preferred embodiments, the following C-terminal TRP-PLIK2deletion polypeptides are encompassed by the present invention:M1-L2017, M1-Q2016, M1-M2015, M1-D2014, M1-D2013, M1-E2012, M1-P2011,M1-S2010, M1-N2009, M1-R2008, M1-G2007, M1-T2006, M1-E2005, M1-R2004,M1-A2003, M1-P2002, M1-P2001, M1-E2000, M1-E1999, M1-A1998, M1-S1997,M1-E1996, M1-11995, M1-K1994, M1-11993, M1-E1992, M1-L1991, M1-G1990,M1-F1989, M1-T1988, M1-S1987, M1-N1986, M1-11985, M1-R1984, M1-E1983,M1-P1982, M1-S1981, M1-Y1980, M1-D1979, M1-N1978, M1-R1977, M1-K1976,M1-LI975, M1-D1974, M1-P1973, M1-L1972, M1-K1971, M1-L1970, M1-K1969,M1-R1968, M1-C1967, M1-C1966, M1-S1965, M1-N1964, M1-C1963, M1-H1962,M1-H1961, M1-K1960, M1-A1959, M1-I1958, M1-F1957, M1-N1956, M1-R1955,M1-I1954, M1-A1953, M1-D1952, M1-E1951, M1-G1950, M1-L1949, M1-N1948,M1-A1947, M1-P1946, M1-G1945, M1-F1944, M1-V1943, M1-M1942, M1-G1941,M1-R1940, M1-S1939, M1-Q1938, M1-K1937, M1-V1936, M1-E1935, M1-P1934,M1-K1933, M1-I1932, M1-V1931, M1-S1930, M1-P1929, M1-D1928, M1-T1927,M1-L1926, M1-N1925, M1-E1924, M1-G1923, M1-V1922, M1-G1921, M1-Q1920,M1-L1919, M1-D1918, M1-L1917, M1-V1916, M1-L1915, M1-L1914, M1-E1913,M1-G1912, M1-R1911, M1-T1910, M1-Y1909, M1-E1908, M1-Y1907, M1-T1906,M1-W1905, M1-H1904, M1-S1903, M1-F1902, M1-A1901, M1-L1900, M1-M1899,M1-L1898, M1-E1897, M1-E1896, M1-L1895, M1-T1894, M1-N1893, M1-T1892,M1-P1891, M1-T1890, M1-I1889, M1-E1888, M1-D1887, M1-G1886, M1-N1885,M1-N1884, M1-N1883, M1-N1882, M1-Y1881, M1-K1880, M1-R1879, M1-F1878,M1-E1877, M1-G1876, M1-T1875, M1-M1874, M1-Y1873, M1-K1872, M1-E1871,M1-I1870, M1-T1869, M1-L1868, M1-W1867, M1-Q1866, M1-N1865, M1-A1864,M1-S1863, M1-H1862, M1-C1861, M1-Y1860, M1-I1859, M1-L1858, M1-F1857,M1-V1856, M1-E1855, M1-L1854, M1-F1853, M1-R1852, M1-P1851, M1-T1850,M1-Y1849, M1-P1848, M1-I1847, M1-T1846, M1-Q1845, M1-P1844, M1-K1843,M1-V1842, M1-Q1841, M1-N1840, M1-F1839, M1-T1838, M1-Y1837, M1-I1836,M1-L1835, M1-K1834, M1-Q1833, M1-A1832, M1-A1831, M1-R1830, M1-Q1829,M1-Q1828, M1-Q1827, M1-I1826, M1-E1825, M1-R1824, M1-L1823, M1-C1822,M1-L1821, M1-H1820, M1-L1819, M1-V1818, M1-T1817, M1-S1816, M1-E1815,M1-Q1814, M1-F1813, M1-I1812, M1-K1811, M1-H1810, M1-W1809, M1-T1808,M1-R1807, M1-V1806, M1-V1805, M1-E1804, M1-P1803, M1-L1802, M1-F1801,M1-S1800, M1-K1799, M1-V1798, M1-I1797, M1-F1796, M1-V1795, M1-Q1794,M1-G1793, M1-P1792, M1-K1791, M1-L1790, M1-I1789, M1-D1788, M1-D1787,M1-E1786, M1-S1785, M1-W1784, M1-T1783, M1-S1782, M1-V1781, M1-V1780,M1-R1779, M1-M1778, M1-A1777, M1-K1776, M1-R1775, M1-L1774, M1-G1773,M1-G1772, M1-D1771, M1-M1770, M1-E1769, M1-E1768, M1-R1767, M1-S1766,M1-L1765, M1-V1764, M1-Q1763, M1-I1762, M1-Ml761, M1-A1760, M1-A1759,M1-R1758, M1-G1757, M1-R1756, M1-Q1755, M1-S1754, M1-W1753, M1-S1752,M1-S1751, M1-M1750, M1-S1749, M1-K1748, M1-D1747, M1-L1746, M1-N1745,M1-L1744, M1-P1743, M1-S1742, M1-S1741, M1-E1740, M1-E1739, M1-L1738,M1-R1737, M1-Y1736, M1-V1735, M1-T1734, M1-I1733, M1-E1732, M1-E1731,M1-G1730, M1-A1729, M1-F1728, M1-L1727, M1-Q1726, M1-V1725, M1-P1724,M1-T1723, M1-F1722, M1-P1721, M1-I1720, M1-T1719, M1-Q1718, M1-S1717,M1-L1716, M1-R1715, M1-M1714, M1-L1713, M1-N1712, M1-N1711, M1-R1710,M1-E1709, M1-I1708, M1-A1707, M1-S1706, M1-Y1705, M1-H1704, M1-H1703,M1-H1702, M1-P1701, M1-E1700, M1-Q1699, M1-P1698, M1-S1697, M1-K1696,M1-L1695, M1-S1694, M1-A1693, M1-S1692, M1-I1691, M1-K1690, M1-D1689,M1-V1688, M1-G1687, M1-I1686, M1-S1685, M1-S1684, M1-K1683, M1-L1682,M1-L1681, M1-S1680, M1-N1679, M1-R1678, M1-N1677, M1-L1676, M1-N1675,M1-T1674, M1-S1673, M1-R1672, M1-S1671, M1-N1670, M1-W1669, M1-L1668,M1-S1667, M1-N1666, M1-K1665, M1-S1664, M1-L1663, M1-D1662, M1-E1661,M1-Q1660, M1-S1659, M1-Q1658, M1-K1657, M1-L1656, M1-Y1655, M1-D1654,M1-S1653, M1-I1652, M1-Q1651, M1-I1650, M1-A1649, M1-C1648, M1-Q1647,M1-G1646, M1-I1645, M1-E1644, M1-K1643, M1-T1642, M1-K1641, M1-M1640,M1-K1639, M1-Q1638, M1-H1637, M1-I1636, M1-Y1635, M1-P1634, M1-E1633,M1-V1632, M1-G1631, M1-T1630, M1-H1629, M1-S1628, M1-F1627, M1-K1626,M1-S1625, M1-V1624, M1-T1623, M1-F1622, M1-W1621, M1-N1620, M1-K1619,M1-S1618, M1-Y1617, M1-E1616, M1-E1615, M1-E1614, M1-S1613, M1-I1612,M1-S1611, M1-N1610, M1-E1609, M1-G1608, M1-P1607, M1-E1606, M1-P1605,M1-N1604, M1-L1603, M1-Q1602, M1-D1601, M1-S1600, M1-Q1599, M1-S1598,M1-C1597, M1-A1596, M1-N1595, M1-V1594, M1-T1593, M1-I1592, M1-I1591,M1-P1590, M1-V1589, M1-Q1588, M1-L1587, M1-G1586, M1-Q1585, M1-T1584,M1-N1583, M1-K1582, M1-K1581, M1-K1580, M1-K1579, M1-S1578, M1-L1577,M1-R1576, M1-R1575, M1-D1574, M1-K1573, M1-T1572, M1-L1571, M1-M1570,M1-K1569, M1-A1568, M1-K1567, M1-V1566, M1-W1565, M1-A1564, M1-G1563,M1-Q1562, M1-G1561, M1-I1560, M1-E1559, M1-S1558, M1-S1557, M1-G1556,M1-S1555, M1-L1554, M1-N1553, M1-K1552, M1-I1551, M1-K1550, M1-C1549,M1-I1548, M1-K1547, M1-M1546, M1-L1545, M1-K1544, M1-E1543, M1-E1542,M1-K1541, M1-H1540, M1-F1539, M1-R1538, M1-F1537, M1-S1536, M1-H1535,M1-S1534, M1-R1533, M1-A1532, M1-F1531, M1-P1530, M1-R1529, M1-Y1528,M1-R1527, M1-R1526, M1-L1525, M1-P1524, M1-N1523, M1-I1522, M1-W1521,M1-F1520, M1-S1519, M1-T1518, M1-N1517, M1-P1516, M1-Q1515, M1-L1514,M1-W1513, M1-P1512, M1-G1511, M1-V1510, M1-E1509, M1-S1508, M1-C1507,M1-E1506, M1-S1505, M1-S1504, M1-Q1503, M1-A1502, M1-S1501, M1-R1500,M1-T1499, M1-S1498, M1-N1497, M1-D1496, M1-S1495, M1-L1494, M1-S1493,M1-S1492, M1-D1491, M1-Q1490, M1-A1489, M1-Q1488, M1-K1487, M1-Q1486,M1-H1485, M1-Q1484, M1-E1483, M1-S1482, M1-R1481, M1-S1480, M1-S1479,M1-D1478, M1-S1477, M1-D1476, M1-C1475, M1-T1474, M1-S1473, M1-P1472,M1-L1471, M1-C1470, M1-T1469, M1-Q1468, M1-W1467, M1-K1466, M1-K1465,M1-K1464, M1-I1463, M1-S1462, M1-F1461, M1-V1460, M1-G1459, M1-T1458,M1-E1457, M1-D1456, M1-G1455, M1-E1454, M1-S1453, M1-F1452, M1-A1451,M1-W1450, M1-N1449, M1-V1448, M1-Y1447, M1-G1446, M1-G1445, M1-G1444,M1-T1443, M1-Q1442, M1-M1441, M1-I1440, M1-K1439, M1-A1438, M1-Q1437,M1-S1436, M1-L1435, M1-P1434, M1-S1433, M1-S1432, M1-M1431, M1-T1430,M1-M1429, M1-P1428, M1-E1427, M1-P1426, M1-T1425, M1-C1424, M1-S1423,M1-L1422, M1-T1421, M1-P1420, M1-L1419, M1-V1418, M1-Q1417, M1-E1416,M1-A1415, M1-K1414, M1-D1413, M1-Q1412, M1-G1411, M1-D1410, M1-L1409,M1-L1408, M1-H1407, M1-A1406, M1-I1405, M1-P1404, M1-E1403, M1-H1402,M1-K1401, M1-E1400, M1-K1399, M1-P1398, M1-E1397, M1-D1396, M1-V1395,M1-S1394, M1-A1393, M1-W1392, M1-D1391, M1-S1390, M1-V1389, M1-V1388,M1-P1387, M1-T1386, M1-Q1385, M1-G1384, M1-T1383, M1-L1382, M1-H1381,M1-V1380, M1-L1379, M1-V1378, M1-E1377, M1-T1376, M1-Q1375, M1-I1374,M1-D1373, M1-Q1372, M1-E1371, M1-T1370, M1-A1369, M1-L1368, M1-V1367,M1-D1366, M1-P1365, M1-V1364, M1-S1363, M1-P1362, M1-R1361, M1-S1360,M1-L1359, M1-P1358, M1-L1357, M1-V1356, M1-T1355, M1-E1354, M1-A1353,M1-S1352, M1-F1351, M1-P1350, M1-V1349, M1-R1348, M1-K1347, M1-L1346,M1-N1345, M1-S1344, M1-P1343, M1-V1342, M1-L1341, M1-L1340, M1-F1339,M1-Q1338, M1-G1337, M1-Y1336, M1-K1335, M1-S1334, M1-H1333, M1-A1332,M1-Q1331, M1-R1330, M1-N1329, M1-P1328, M1-S1327, M1-V1326, M1-G1325,M1-S1324, M1-V1323, M1-V1322, M1-I1321, M1-S1320, M1-S1319, M1-Q1318,M1-T1317, M1-E1316, M1-Q1315, M1-R1314, M1-E1313, M1-Q1312, M1-D1311,M1-N1310, M1-R1309, M1-V1308, M1-N1307, M1-T1306, M1-A1305, M1-E1304,M1-R1303, M1-K1302, M1-S1301, M1-N1300, M1-T1299, M1-I1298, M1-El297,M1-L1296, M1-L1295, M1-A1294, M1-G1293, M1-R1292, M1-Q1291, M1-Vl290,M1-R1289, M1-P1288, M1-P1287, M1-H1286, M1-R1285, M1-G1284, M1-G1283,M1-A1282, M1-L1281, M1-S1280, M1-R1279, M1-L1278, M1-L1277, M1-Sl276,M1-S1275, M1-P1274, M1-M1273, M1-S1272, M1-Y1271, M1-Y1270, M1-Q1269,M1-Y1268, M1-K1267, M1-K1266, M1-E1265, M1-G1264, M1-Al263, M1-I1262,M1-E1261, M1-M1260, M1-S1259, M1-G1258, M1-L1257, M1-V1256, M1-E1255,M1-A1254, M1-C1253, M1-I1252, M1-V1251, M1-N1250, M1-S1249, M1-W1248,M1-S1247, M1-H1246, M1-P1245, M1-L1244, M1-K1243, M1-K1242, M1-C1241,M1-T1240, M1-S1239, M1-H1238, M1-K1237, M1-R1236, M1-K1235, M1-A1234,M1-L1233, M1-L1232, M1-A1231, M1-E1230, M1-D1229, M1-E1228, M1-Q1227,M1-L1226, M1-T1225, M1-D1224, M1-V1223, M1-A1222, M1-S1221, M1-L1220,M1-V1219, M1-K1218, M1-L1217, M1-T1216, M1-D1215, M1-V1214, M1-T1213,M1-L1212, M1-A1211, M1-S1210, M1-L1209, M1-D1208, M1-Q1207, M1-L1206,M1-H1205, M1-G1204, M1-V1203, M1-Q1202, M1-S1200, M1-D1200, M1-L1199,M1-S1198, M1-L1197, M1-L1196, M1-S1195, M1-D1194, M1-K1193, M1-I1192,M1-F1191, M1-S1190, M1-VI189, M1-K1188, M1-E1187, M1-N1186, M1-M1185,M1-E1184, M1-Ki183, M1-L1182, M1-Q1181, M1-F1180, M1-Y1179, M1-M1178,M1-E1177, M1-T1176, M1-VI175, M1-R1174, M1-E1173, M1-S1172, M1-T1171,M1-V1170, M1-R1169, M1-I1168, M1-R1167, M1-E1166, M1-E1165, M1-C1164,M1-S1163, M1-C1162, M1-N1161, M1-V1160, M1-D1159, M1-E1158, M1-M1157,M1-K1156, M1-E1155, M1-H1154, M1-F1153, M1-Y1152, M1-K1151, M1-E1150,M1-V1149, M1-C1148, M1-Q1147, M1-E1146, M1-E1145, M1-F1144, M1-D1143,M1-H1142, M1-L1141, M1-K1140, M1-K1139, M1-L1138, M1-D1137, M1-E1136,M1-K1135, M1-S1134, M1-L1133, M1-Y1132, M1-L1131, M1-K1130, M1-L1129,M1-G1128, M1-V1127, M1-D1126, M1-G1125, M1-E1124, M1-E1123, M1-Q1122,M1-D1121, M1-H1120, M1-P1119, M1-A1118, M1-R1117, M1-H1116, M1-C1115,M1-C1114, M1-L1113, M1-R1112, M1-R1111, M1-L1110, M1-L1109, M1-L1108,M1-G1107, M1-V1106, M1-H1105, M1-S1104, M1-L1103, M1-L1102, M1-I1101,M1-L1100, M1-P1099, M1-P1098, M1-P1097, M1-L1096, M1-W1095, M1-P1094,M1-K1093, M1-E1092, M1-H1091, M1-Y1090, M1-T1089, M1-M1088, M1-I1087,M1-Y1086, M1-R1085, M1-Y1084, M1-R1083, M1-N1082, M1-Y1081, M1-K1080,M1-W1079, M1-L1078, M1-N1077, M1-N1076, M1-S1075, M1-I1074, M1-S1073,M1-E1072, M1-M1071, M1-D1070, M1-L1069, M1-Y1068, M1-V1067, M1-N1066,M1-N1065, M1-F1064, M1-F1063, M1-A1062, M1-I1061, M1-L1060, M1-L1059,M1-N1058, M1-V1057, M1-M1056, M1-I1055, M1-I1054, M1-Y1053, M1-Q1052,M1-V1051, M1-F1050, M1-L1049, M1-Y1048, M1-V1047, M1-A1046, M1-Q1045,M1-L1044, M1-F1043, M1-P1042, M1-T1041, M1-L1040, M1-F1039, M1-S1038,M1-G1037, M1-P1036, M1-P1035, M1-C1034, M1-S1033, M1-P1032, M1-Q1031,M1-S1030, M1-S1029, M1-C1028, M1-V1027, M1-D1026, M1-I1025, M1-E1024,M1-G1023, M1-A1022, M1-Y1021, M1-V1020, M1-E1019, M1-G1018, M1-Y1017,M1-I1016, M1-M1015, M1-W1014, M1-Y1013, M1-P1012, M1-E1011, M1-F1010,M1-V1009, M1-I1008, M1-D1007, M1-R1006, M1-A1005, M1-L1004, M1-S1003,M1-W1002, M1-S1001, M1-P1000, M1-P999, M1-E998, M1-K997, M1-P996,M1-S995, M1-L994, M1-1993, M1-A992, M1-K991, M1-R990, M1-A989, M1-V988,M1-G987, M1-F986, M1-S985, M1-L984, M1-L983, M1-V982, M1-1981, M1-A980,M1-M979, M1-1978, M1-1977, M1-V976, M1-1975, M1-Y974, M1-F973, M1-M972,M1-N971, M1-A970, M1-T969, M1-M968, M1-K967, M1-A966, M1-1965, M1-M964,M1-T963, M1-V962, M1-Y961, M1-P960, M1-G959, M1-A958, M1-H957, M1-Q956,M1-N955, M1-V954, M1-A953, M1-F952, M1-F951, M1-D950, M1-L949, M1-L948,M1-R947, M1-S946, M1-F945, M1-W944, M1-F943, M1-1942, M1-1941, M1-D940,M1-1939, M1-C938, M1-Y937, M1-1936, M1-L935, M1-R934, M1-G933, M1-A932,M1-T931, M1-H930, M1-F929, M1-P928, M1-P927, M1-D926, M1-G925, M1-W924,M1-R923, M1-L922, M1-V921, M1-F920, M1-G919, M1-A918, M1-S917, M1-F916,M1-L915, M1-G914, M1-1913, M1-A912, M1-V911, M1-T910, M1-E909, M1-T908,M1-L907, M1-N906, M1-W905, M1-Y904, M1-E903, M1-S902, M1-1901, M1-W900,M1-V899, M1-K898, M1-V897, M1-K896, M1-Q895, M1-T894, M1-F893, M1-K892,M1-G891, M1-P890, M1-E889, M1-S888, M1-1887, M1-C886, M1-1885, M1-E884,M1-R883, M1-V882, M1-V881, M1-E880, M1-1879, M1-A878, M1-N877, M1-T876,M1-F875, M1-1874, M1-Y873, M1-1872, M1-S871, M1-V870, M1-L869, M1-W868,M1-E867, M1-Q866, M1-V865, M1-S864, M1-P863, M1-Q862, M1-P861, M1-Q860,M1-M859, M1-E858, M1-V857, M1-L856, M1-V855, M1-T854, M1-Y853, M1-T852,M1-F851, M1-L850, M1-M849, M1-L848, M1-F847, M1-A846, M1-L845, M1-Y844,M1-A843, M1-M842, M1-T841, M1-Y840, M1-F839, M1-W838, M1-F837, M1-K836,M1-V835, M1-1834, M1-P833, M1-A832, M1-S831, M1-Y830, M1-F829, M1-E828,M1-Y827, M1-V826, M1-K825, M1-R824, M1-T823, M1-W822, M1-P821, M1-L820,M1-H819, M1-Q818, M1-H817, M1-G816, M1-S815, M1-E814, M1-L813, M1-G812,M1-F811, M1-H810, M1-Q809, M1-N808, M1-E807, M1-D806, M1-L805, M1-K804,M1-E803, M1-D802, M1-H801, M1-G800, M1-R799, M1-E798, M1-L797, M1-D796,M1-Y795, M1-E794, M1-K793, M1-V792, M1-S791, M1-A790, M1-S789, M1-E788,M1-K787, M1-S786, M1-S785, M1-S784, M1-A783, M1-N782, M1-Q781, M1-D780,M1-S779, M1-Y778, M1-Y777, M1-W776, M1-M775, M1-F774, M1-Q773, M1-F772,M1-D771, M1-Q770, M1-S769, M1-Q768, M1-P767, M1-V766, M1-H765, M1-S764,M1-M763, M1-E762, M1-A761, M1-K760, M1-S759, M1-K758, M1-F757, M1-E756,M1-L755, M1-T754, M1-L753, M1-1752, M1-T751, M1-P750, M1-P749, M1-L748,M1-1747, M1-1746, M1-S745, M1-1744, M1-1743, M1-1742, M1-K741, M1-L740,M1-W739, M1-S738, M1-N737, M1-K736, M1-R735, M1-M734, M1-K733, M1-L732,M1-R731, M1-G730, M1-M729, M1-W728, M1-M727, M1-D726, M1-T725, M1-L724,M1-L723, M1-M722, M1-Q721, M1-T720, M1-C719, M1-T718, M1-H717, M1-S716,M1-V715, M1-F714, M1-P713, M1-R712, M1-L711, M1-G710, M1-G709, M1-S708,M1-V707, M1-A706, M1-L705, M1-K704, M1-L703, M1-C702, M1-T701, M1-S700,M1-N699, M1-S698, M1-W697, M1-N696, M1-R695, M1-L694, M1-E693, M1-Y692,M1-T691, M1-L690, M1-L689, M1-T688, M1-M687, M1-A686, M1-M685, M1-R684,M1-E683, M1-N682, M1-Q681, M1-K680, M1-F679, M1-A678, M1-K677, M1-E676,M1-L675, M1-L674, M1-D673, M1-L672, M1-A671, M1-L670, M1-Q669, M1-G668,M1-F667, M1-Q666, M1-K665, M1-S664, M1-Y663, M1-N662, M1-K661, M1-L660,M1-E659, M1-E658, M1-S657, M1-A656, M1-D655, M1-D654, M1-V653, M1-M652,M1-H651, M1-S650, M1-E649, M1-K648, M1-A647, M1-E646, M1-H645, M1-A644,M1-M643, M1-A642, M1-R641, M1-Y640, M1-L639, M1-1638, M1-C637, M1-A636,M1-1635, M1-V634, M1-A633, M1-K632, M1-V631, M1-T630, M1-A629, M1-E628,M1-E627, M1-G626, M1-H625, M1-Q624, M1-W623, M1-F622, M1-F621, M1-M620,M1-A619, M1-M618, M1-K617, M1-Q616, M1-R615, M1-K614, M1-M613, M1-L612,M1-V611, M1-A610, M1-W609, M1-V608, M1-L607, M1-L606, M1-D605, M1-N604,M1-Y603, M1-P602, M1-Y601, M1-L600, M1-F599, M1-G598, M1-T597, M1-S596,M1-E595, M1-P594, M1-D593, M1-D592, M1-S591, M1-V590, M1-N589, M1-Q588,M1-E587, M1-K586, M1-S585, M1-K584, M1-K583, M1-R582, M1-S581, M1-K580,M1-H579, M1-L578, M1-V577, M1-1576, M1-S575, M1-K574, M1-E573, M1-K572,M1-F571, M1-K570, M1-Y569, M1-P568, M1-Q567, M1-A566, M1-T565, M1-R564,M1-1563, M1-F562, M1-Q561, M1-S560, M1-H559, M1-L558, M1-T557, M1-S556,M1-E555, M1-A554, M1-S553, M1-E552, M1-N551, M1-R550, M1-N549, M1-G548,M1-S547, M1-S546, M1-H545, M1-R544, M1-Q543, M1-H542, M1-K541, M1-Y540,M1-K539, M1-R538, M1-Y537, M1-L536, M1-N535, M1-N534, M1-Y533, M1-L532,M1-A531, M1-R530, M1-F529, M1-H528, M1-K527, M1-R526, M1-T525, M1-Y524,M1-N523, M1-S522, M1-R521, M1-Y520, M1-A519, M1-R518, M1-G517, M1-1516,M1-L515, M1-Y514, M1-E513, M1-V512, M1-V511, M1-L510, M1-G509, M1-1508,M1-D507, M1-1506, M1-L505, M1-T504, M1-1503, M1-R502, M1-Y501, M1-G500,M1-S499, M1-L498, M1-L497, M1-T496, M1-H495, M1-Q494, M1-K493, M1-V492,M1-D491, M1-Q490, M1-V489, M1-L488, M1-H487, M1-H486, M1-ILA85, M1-L484,M1-T483, M1-N482, M1-T481, M1-P480, M1-G479, M1-Q478, M1-K477, M1-T476,M1-N475, M1-Y474, M1-L473, M1-E472, M1-E471, M1-L470, M1-R469, M1-P468,M1-1467, M1-T466, M1-L465, M1-F464, M1-R463, M1-H462, M1-L461, M1-N460,M1-V459, M1-G458, M1-Y457, M1-E456, M1-1455, M1-L454, M1-IA53, M1-K452,M1-V451, M1-F450, M1-D449, M1-V448, M1-R447, M1-D446, M1-M445, M1-V444,M1-L443, M1-A442, M1-D441, M1-S440, M1-M439, M1-A438, M1-Q437, M1-E436,M1-L435, M1-A434, M1-D433, M1-P432, M1-K431, M1-W430, M1-H429, M1-Q428,M1-E427, M1-Y426, M1-1425, M1-L424, M1-1423, M1-H422, M1-K421, M1-K420,M1-A419, M1-1418, M1-D417, M1-V416, M1-R415, M1-D414, M1-W413, M1-A412,M1-M411, M1-A410, M1-L409, M1-N408, M1-L407, M1-Q406, M1-E405, M1-S404,M1-A403, M1-S402, M1-L401, M1-N400, M1-T399, M1-G398, M1-K397, M1-L396,M1-L395, M1-A394, M1-T393, M1-L392, M1-1391, M1-A390, M1-L389, M1-D388,M1-L387, M1-D386, M1-Q385, M1-Q384, M1-E383, M1-E382, M1-S381, M1-D380,M1-A379, M1-D378, M1-F377, M1-1376, M1-T375, M1-1374, M1-C373, M1-D372,M1-R371, M1-H370, M1-V369, M1-M368, M1-C367, M1-E366, M1-M365, M1-L364,M1-1363, M1-Q362, M1-F361, M1-L360, M1-H359, M1-K358, M1-S357, M1-Q356,M1-K355, M1-L354, M1-S353, M1-F352, M1-N351, M1-F350, M1-T349, M1-N348,M1-Q347, M1-1346, M1-M345, M1-C344, M1-1343, M1-1342, M1-E341, M1-E340,M1-K339, M1-V338, M1-Q337, M1-P336, M1-R335, M1-L334, M1-M333, M1-G332,M1-E331, M1-D330, M1-A329, M1-L328, M1-H327, M1-K326, M1-H325, M1-T324,M1-F323, M1-A322, M1-L321, M1-L320, M1-D319, M1-A318, M1-A317, M1-R316,M1-G315, M1-T314, M1-G313, M1-E312, M1-C311, M1-V310, M1-V309, M1-V308,M1-P307, M1-D306, M1-K305, M1-D304, M1-K303, M1-V302, M1-T301, M1-E300,M1-W299, M1-V298, M1-S297, M1-L296, M1-1295, M1-V294, M1-N293, M1-P292,M1-G291, M1-G290, M1-E289, M1-V288, M1-V287, M1-L286, M1-G285, M1-V284,M1-V283, M1-P282, M1-V281, M1-G280, M1-Q279, M1-R278, M1-S277, M1-R276,M1-C275, M1-H274, M1-1273, M1-K272, M1-Q271, M1-L270, M1-S269, M1-L268,M1-Y267, M1-K266, M1-E265, M1-L264, M1-N263, M1-R262, M1-R261, M1-L260,M1-K259, M1-M258, M1-E257, M1-N256, M1-G255, M1-Y254, M1-K253, M1-G252,M1-V251, M1-T250, M1-G249, M1-D248, M1-D247, M1-S246, M1-L245, M1-1244,M1-F243, M1-H242, M1-S241, M1-H240, M1-M239, M1-S238, M1-N237, M1-L236,M1-T235, M1-T234, M1-L233, M1-K232, M1-S231, M1-L230, M1-P229, M1-N228,M1-D227, M1-L226, M1-T225, M1-Q224, M1-Y223, M1-L222, M1-C221, M1-V220,M1-V219, M1-D218, M1-K217, M1-G216, M1-1215, M1-L214, M1-D213, M1-R212,M1-Q211, M1-N210, M1-E209, M1-1208, M1-V207, M1-G206, M1-W205, M1-P204,M1-P203, M1-1202, M1-G201, M1-V200, M1-T199, M1-W198, M1-1197, M1-K196,M1-R195, M1-L194, M1-S193, M1-H192, M1-S191, M1-S190, M1-H189, M1-S188,M1-K187, M1-L186, M1-A185, M1-D184, M1-G183, M1-V182, M1-H181, M1-K180,M1-S179, M1-V178, M1-G177, M1-T176, M1-N175, M1-1174, M1-G173, M1-E172,M1-T171, M1-1170, M1-I169, M1-W168, M1-A167, M1-G166, M1-T165, M1-T164,M1-E163, M1-A162, M1-A161, M1-K160, M1-V159, M1-L158, M1-G157, M1-Q156,M1-S155, M1-F154, M1-I153, M1-El52, M1-K151, M1-F150, M1-K149, M1-S148,M1-P147, M1-M146, M1-T145, M1-F144, M1-N143, M1-Q142, M1-1141, M1-G140,M1-G139, M1-H138, M1-V137, M1-S136, M1-I135, M1-V134, M1-L133, M1-K132,M1-P131, M1-L130, M1-E129, M1-M128, M1-K127, M1-W126, M1-E125, M1-K124,M1-L123, M1-M122, M1-L121, M1-H120, M1-L119, M1-L118, M1-H117, M1-D116,M1-L115, M1-K114, M1-T113, M1-D112, M1-Y111, M1-SI 10, M1-T109, M1-R108,M1-I107, M1-Y106, M1-K105, M1-A104, M1-H103, M1-H102, M1-T101, M1-H100,M1-E99, M1-G98, M1-D97, M1-Q96, M1-F95, M1-N94, M 1-193, M1-T92, M1-G91,M1-F90, M1-T89, M1-D88, M1-T87, M1-P86, M1-S85, M1-K84, M1-T83, M1-T82,M1-H81, M1-K80, M1-E79, M1-V78, M1-S77, M1-W76, M1-Q75, M1-E74, M1-S73,M1-E72, M1-K71, M1-G70, M1-K69, M1-A68, M1-A67, M1-S66, M1-165, M1-T64,M1-W63, M1-S62, M1-Y61, M1-D60, M1-159, M1-G58, M1-A57, M1-H56, M1-D55,M1-G54, M1-153, M1-L52, M1-R51, M1-G50, M1-C49, M1-Y48, M1-C47, M I-R46,M1-145, M1-L44, M1-N43, M1-Q42, M1-C41, M1-V40, M1-Q39, M1-C38, M1-V37,M1-P36, M1-T35, M1-C34, M1-R33, M1-H32, M1-P31, M1-N30, M1-K29, M1-S28,M1-S27, M1-P26, M1-125, M1-I24, M1-T23, M1-S22, M1-C21, M1-E20, M1-R19,M1-K18, M1-D17, M1-F16, M1-V15, M1-G14, M1-K13, M1-I12, M1-W11, M1-S1O,M1-K9, M1-Q8, and/or M1-S7 of SEQ ID NO:2. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these C-terminal TRP-PLIK2 deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0185] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the TRP-PLIK2 polypeptide (e.g., any combination ofboth N- and C- terminal TRP-PLIK2 polypeptide deletions) of SEQ ID NO:2.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of TRP-PLIK2 (SEQ ID NO:2), and where CX refersto any C-terminal deletion polypeptide amino acid of TRP-PLIK2 (SEQ IDNO:2). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of these polypeptides asan immunogenic and/or antigenic epitope as described elsewhere herein.

[0186] The TRP-PLIK2 polypeptides of the present invention weredetermined to comprise several phosphorylation sites based upon theMotif algorithm (Genetics Computer Group, Inc.). The phosphorylation ofsuch sites may regulate some biological activity of the TRP-PLIK2polypeptide. For example, phosphorylation at specific sites may beinvolved in regulating the proteins ability to associate or bind toother molecules (e.g., proteins, ligands, substrates, DNA, etc.). In thepresent case, phosphorylation may modulate the ability of the TRP-PLIK2polypeptide to associate with other potassium channel alpha subunits,beta subunits, or its ability to modulate potassium channel function.

[0187] The TRP-PLIK2 polypeptide was predicted to comprise twenty ninePKC phosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. . . .260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0188] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: ILSKSQKSWIKG (SEQID NO:51), STIIPSSKNPHRC (SEQ ID NO:52), SVEKHTTKSPTDT (SEQ ID NO:53),SHSSHSLRKIWTV (SEQ ID NO:54), LSVWETVKDKDPV (SEQ ID NO:55),VVCEGTGRAADLL (SEQ ID NO:56), DLLAFTHKHLADE (SEQ ID NO:57),NTFNFSLKQSKHL (SEQ ID NO:58), YRSNYTRKHFRAL (SEQ ID NO:59),IVLHKSRKKSKEQ (SEQ ID NO:60), HGEEATVKAVIAC (SEQ ID NO:61),DQNASSSKESASV (SEQ ID NO:62), SKESASVKEYDLE (SEQ ID NO:63),QHLPWTRKVYEFY (SEQ ID NO:64), EPGKFTQKVKVWI (SEQ ID NO:65),RKAILSPKEPPSW (SEQ ID NO:66), RIRVTSERVTEMY (SEQ ID NO:67),ALTVDTLKVLSAV (SEQ ID NO:68), KRKHSTCKKLPHS (SEQ ID NO:69),LEITNSKREATNV (SEQ ID NO:70), ETGVFSIKKKWQT (SEQ ID NO:71),TCDSDSSRSEQHQ (SEQ ID NO:72), SLSDNSTRSAQSS (SEQ ID NO:73),FARSHSFRFHKEE (SEQ ID NO:74), KDRRLSKKKKNTQ (SEQ ID NO:75),DKISASLKSPQEP (SEQ ID NO:76), SMSSWSQRGRAAM (SEQ ID NO:77),QTIPYTPRFLEVF (SEQ ID NO:78), and/or PPARETGRNSPED (SEQ ID NO:79).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these TRP-PLIK2 PKCphosphorylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0189] The present invention also encompasses immunogenic and/orantigenic epitopes of the TRP-PLIK2 polypeptide.

[0190] The TRP-PLIK2 polypeptide has been shown to comprise seventeenglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0191] Asparagine phosphorylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D.D., Lennarz W.J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. . .. 265:11397-I1404(1990).

[0192] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:HGGIQNFTMPSKFK (SEQ ID NO:80), IQNTFNFSLKQSKH (SEQ ID NO:81),LLKGTNLSASEQLN (SEQ ID NO:82), RAYRSNYTRKHFRA (SEQ ID NO:83),SSGNRNESAESTLH (SEQ ID NO:84), KSKEQNVSDDPEST (SEQ ID NO:85),SEELKNYSKQFGQL (SEQ ID NO:86), TYELRNWSNSTCLK (SEQ ID NO:87),LRNWSNSTCLKLAV (SEQ ID NO:88), YYSDQNASSSKESA (SEQ ID NO:89),ISEYWNLTETVAIG (SEQ ID NO:90), KMEDVNCSCEERIR (SEQ ID NO:91),SSLSDNSTRSAQSS (SEQ ID NO:92), PWLQPNTSFWINPL (SEQ ID NO:93),ICKIKNLSGSSEIG (SEQ ID NO:94), QGVGENLTDPSVIK (SEQ ID NO:95), and/orSPERINSTFGLEIK (SEQ ID NO:96). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these TRP-PLIK2 asparagine glycosylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0193] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 6040 ofSEQ ID NO:1, b is an integer between 15 to 6054, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:1,and where b is greater than or equal to a+14.

[0194] In one embodiment, a TRP-PLIK2 polypeptide comprises a portion ofthe amino sequence depicted in FISS. 1A-G. In another embodiment, aTRP-PLIK2 polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the amino sequencedepicted in FIGS. 1A-G. In further embodiments, the following TRP-PLIK2polypeptide fragments are specifically excluded from the presentinvention: VKDKDPVVVCEGTGRAADLLAFTHKHLADEGMLRPQVKEEIICMIQNTFNFSLKQSKHLFQILMECMVHRDCITIFDADSEEQQDLDLAILTALLKGTNLSASEQLNLAMAWDRVDIAKKHILIYEQHWKPDALEQAMSDALVMDRVDFVKLLIEYGVNLHRFLTIPRLEELYNTKQGPTNTLLHHLVQDVKQHTLLSGYRITL (SEQ ID NO:312);KSPTDTFGTINFQDGEH (SEQ ID NO:313); DHLLHLMLKEWMELPKLVISVHGG (SEQ IDNO:314); FSQGLVKAAETTGAWIITEGIN (SEQ ID NO:315); SLRKIWTVGIPPWGVIENQR(SEQ ID NO:316); TVLHLCLREIQQQRAAQKL (SEQ ID NO:317), MTGEFRKYNNNNGDEI(SEQ ID NO:318), MLAFSHWTYEYTRGELLVLDLQGVGENLTDPSVIK (SEQ ID NO:319),AKHHCNSCCRKLKLPDLKRNDY (SEQ ID NO:320),GKDVVCLYQTLDNPLSKLTTLNSMHSHFILSDDGTVGKYGNEMKLRRNLEKYLSLQKIHCRSRQGVPVVGLVVEGGPNVILSVWETVKDKDPVVVCEGTGRAADLLAFTHKHLADEGMLRPQVKEEIICMIQNTFNFSLKQSKHLFQILMECMVH RDC (SEQ IDNO:321), EYTRGELLVLDLQGVGENLTDPSVIK (SEQ ID NO:322), YPYNDLLVWAVLMKRQ(SEQ ID NO:323), MAMFFWQHGEEATVKAVIA (SEQ ID NO:324),NWSNSTCLKLAVSGGLRPFVSH (SEQ ID NO:325), QMLLTDMWMGRLKMRKNSWLKIIISI (SEQID NO:326), LKPGQVFIVKSFLPEVV (SEQ ID NO:327),KIFQESTVLHLCLREIQQQRAAQKLIYTFNQVKPQTIPYTPRFLEV (SEQ ID NO:328),YCHSANQWLTIEKYMTGEFRKYNNNNGDEI (SEQ ID NO:329),PTNTLEELMLAFSHWTYEYTRGELLVLDLQGVGENLTDP (SEQ ID NO:330),TVLHLCLREIQQQRAAQKL (SEQ ID NO:331), MTGEFRKYNNNNGDEI (SEQ ID NO:332),MLAFSHWTYEYTRGELLVLDLQGVGENLTDPSVIK (SEQ ID NO:333),AKHHCNSCCRKLKLPDLKRNDY (SEQ ID NO:334), and/or EYTRGELLVLDLQGVGENLT (SEQID NO:335).

[0195] Features of the Polypeptide Encoded by Gene No:2

[0196] The polypeptide of this gene provided as SEQ ID NO:4 (FIGS.2A-G), encoded by the polynucleotide sequence according to SEQ ID NO:3(FIGS. 2A-G), and/or encoded by the polynucleotide contained within thedeposited clone, TRP-PLIK2b, has significant homology at the nucleotideand amino acid level to the human channel-kinase 1 protein, also knownas the human CHAK1 or TRP-PLIKB1 protein (CHAK1; Genbank Accession No.gilAF346629; SEQ ID NO:9); ; and the human melastatin 1 protein(Melastatinl; Genbank Accession No. gil3243075; SEQ ID NO:260). Analignment of the TRP-PLIK2b polypeptide with this protein is provided inFIGS. 5A-F.

[0197] The TRP-PLIK2b polypeptide was determined to share 58.1% identityand 66.1% similarity with the human CHAK1 or TRP-PLIKB 1 protein (CHAK1;Genbank Accession No. gilAF346629; SEQ ID NO:9); and was determined toshare 47.2% identity and 57.9% similarity with the human melastatin 1protein (Melastatin1; Genbank Accession No. gil3243075; SEQ ID NO:260)as shown in FIG. 9.

[0198] The CHAK1 protein is believed to represent a member of a newclass of protein kianses referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain.

[0199] The melastatin 1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin 1 could represent a noveltherapeutic in the treatment of melanoa and potentially other cancers.

[0200] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the CHAK1 and melastatin 1 proteins, in addition to,other transient receptor potential channel family members referencedelsewhere herein or otherwise known in the art.

[0201] The TRP-PLIK2b (SEQ ID NO:4) polypeptide represents a novelsplice variant form of the TRP-PLIK2 (SEQ ID NO:2) polypeptide of thepresent invention.

[0202] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theTRP-PLIK2b polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 2A-G. The transmembrane domains arelocated from about amino acid 693 to about amino acid 710 (TM1), fromabout amino acid 787 to about amino acid 804 (TM2), from about aminoacid 861 to about amino acid 873 (TM3), from about amino acid 887 toabout amino acid 904 (TM4), from about amino acid 921 to about aminoacid 938 (TM5), and/or from about amino acid 996 to about amino acid1015 (TM6) of SEQ ID NO:4. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0203] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKIIISIILPPTILTLEF (SEQ ID NO:109), IVKFWFYTMAYLAFLMLF (SEQ ID NO:110),TETVAIGLFSAGF (SEQ ID NO:111), RLIYCIDIIFWFSRLLDF (SEQ ID NO:112),MTANMFYWIIMAIVLLS (SEQ ID NO:113), and/or FLQAVYLFVQYIIMVNLLIA (SEQ IDNO:114). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the TRP-PLIK2btransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0204] In preferred embodiments, the present invention encompasses theuse of N-terminal deletions, C-terminal deletions, or any combination ofN-terminal and C-terminal deletions of any one or more of the TRP-PLIK2bTM1 thru TM6 transmembrane domain polypeptides as antigenic and/orimmunogenic epitopes.

[0205] In preferred embodiments, the present invention also encompassesthe use of N-terminal deletions, C-terminal deletions, or anycombination of N-terminal and C-terminal deletions of any one or more ofthe amino acids intervening (i.e., ion channel extracellular orintracellular loops) the TRP-PLIK2b TM1 thru TM6 transmembrane domainpolypeptides as antigenic and/or immunogenic epitopes.

[0206] The TRP-PLIK2b polypeptide was determined to comprise severalconserved cysteines, at amino acid 21, 34, 38, 41, 47, 49, 311, 367,590, 655, 672, 891, 981, 987, 1067, 1101, 1775, 1814, 1915, 1919, and1920 of SEQ ID No:4 (FIGS. 2A-G). Conservation of cysteines at key aminoacid residues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0207] In confirmation of the TRP-PLIK2b representing a member of thetransient receptor channel family, the TRP-PLIK2b polypeptide wasdetermined to comprise a predicted TRP domain (LWKYNR) located fromabout amino acid 1031 to about amino acid 1036 of SEQ ID NO:4. In thiscontext, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of theabove referenced polypeptide.

[0208] In further confirmation of the TRP-PLIK2b representing a memberof the transient receptor channel family, the TRP-PLIK2b polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 857 to about amino acid 1017 of SEQ ID NO:4.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0209] The TRP-PLIK2b polypeptide was determined to comprise a predictednucleotide binding domain located from about amino acid 1898 to aboutamino acid 1903 of SEQ ID NO:4. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0210] In addition, the TRP-PLIK2b polypeptide was determined tocomprise a predicted zinc finger domain located at about amino acid 1913to about amino acid 1923 of SEQ ID NO:4. In this context, the term“about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids beyond the N-Terminus and/or C-terminus of the above referencedpolypeptide.

[0211] TRP-PLIK2b polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofTRP-PLIK2b by identifying mutations in the TRP-PLIK2b gene usingTRP-PLIK2b sequences as probes or by determining TRP-PLIK2b protein ormRNA expression levels. TRP-PLIK2b polypeptides will be useful inscreens for compounds that affect the activity of the protein.TRP-PLIK2b peptides can also be used for the generation of specificantibodies and as bait in yeast two hybrid screens to find proteins thespecifically interact with TRP-PLIK2b.

[0212] Expression profiling designed to measure the steady state mRNAlevels encoding the TRP-PLIK2 polypeptide showed predominately highexpression levels in bone marrow, kidney, and testis. The TRP-PLIK2polypeptide was also significantly expressioned in liver, and to alesser extent, in small intestine, spinal cord, prostate, uterus, lung,lymph node, stomach, heart, brain, thymus, and pancrease (as shown inFIG. 7). The tissue expression of TRP-PLIK2b may follow the same patternas for the TRP-PLIK2 polypeptide of the present invention.

[0213] Expanded analysis of TRP-PLIK2 expression levels by TaqMan™quantitative PCR (see FIG. 12) confirmed that the TRP-PLIK2 polypeptideis expressed in kidney, colon, and testis (FIG. 7). TRP-PLIK2 mRNA wasexpressed predominately in the lower gastrointestinal tract,specifically the ileum, the rectum, the colon, the jejunum, and to alesser extent in the duodenum and stomach. Significant expression wasobserved in the kidney, particularly in the cortex, followed by themedulla, and to a lesser extent in the testis, pelvis, and bone marrow(mononuclear cells).

[0214] Furthermore, an expanded analysis of TRP-PLIK2 expression levelsin various tumor and normal tissues by TaqMan™ quantitative PCR (seeFIG. 14) showed TRP-PLIK2 mRNA was differentially expressed to thegreatest extent in prostate tumor tissue relative to normal prostatetissue (approximately 20 fold difference). Significant differentalexpression was also observed in the testicular tumor tissue relative tonormal testicular tissue.

[0215] Characterization of the TRP-PLIK2 polypeptide of the presentinvention using antisense oligonucleotides directed against a portion ofthe TRP-PLIK2 encoding sequence led to the determination that it isinvolved in the modulation of the NFkB pathway, either directly orindirectly.

[0216] The upregulation of IkBa due to the downregulation of TRP-PLIK2places this transient receptor potential protein into a signallingpathway potentially involved in apoptotic events. This gives theopportunity to regulate downstream events via the activity of theprotein TRP-PLIK2 with antisense polynucleotides, polypeptides or lowmolecular chemicals with the potential of achieving a therapeutic effectin cancer, autoimmune diseases. In addition to cancer and immunologicaldisorders, NF-kB has significant roles in other diseases (Baldwin, A.S., J. Clin Invest. 107, :3-6 (2001)). NF-kB is a key factor in thepathophysiology of ischemia-reperfusion injury and heart failure (Valen,G., Yan. Z Q, Hansson G K, J. Am. Coll. Cardiol. 38, 307-14 (2001)).Furthermore, NF-kB has been found to be activated in experimental renaldisease (Guijarro C, Egido J., Kidney Int. 59, 415-425 (2001)). AsTRP-PLIK2 is highly expressed in kidney there is the potential of aninvolvement in renal diseases.

[0217] In preferred embodiments, TRP-PLIK2b polynucleotides andpolypeptides, including fragments thereof, are useful for treating,diagnosing, and/or ameliorating proliferative disorders, cancers,ischemia-reperfusion injury, heart failure, immuno compromisedconditions, HIV infection, and renal diseases.

[0218] Moreover, TRP-PLIK2b polynucleotides and polypeptides, includingfragments thereof, are useful for increasing NF-kB activity, increasingapoptotic events, and/or decreasing IkBa expression or activity levels.

[0219] In preferred embodiments, antagonists directed against TRP-PLIK2bare useful for treating, diagnosing, and/or ameliorating autoimmunedisorders, disorders related to hyper immune activity, inflammatoryconditions, disorders related to aberrant acute phase responses,hypercongenital conditions, birth defects, necrotic lesions, wounds,organ transplant rejection, conditions related to organ transplantrejection, disorders related to aberrant signal transduction,proliferating disorders, cancers, HIV, and HIV propagation in cellsinfected with other viruses.

[0220] Moreover, antagonists directed against TRP-PLIK2b are useful fordecreasing NF-kB activity, decreasing apoptotic events, and/orincreasing IkBa expression or activity levels.

[0221] In preferred embodiments, agonists directed against TRP-PLIK2bare useful for treating, diagnosing, and/or ameliorating autoimmunediorders, disorders related to hyper immune activity, hypercongenitalconditions, birth defects, necrotic lesions, wounds, disorders relatedto aberrant signal transduction, immuno compromised conditions, HIVinfection, proliferating disorders, and/or cancers.

[0222] Moreover, agonists directed against TRP-PLIK2b are useful forincreasing NF-kB activity, increasing apoptotic events, and/ordecreasing IkBa expression or activity levels.

[0223] The strong homology to transient receptor potential channels(TRP), combined with the predominate localized expression of theTRP-PLIK2 polypeptide in the lower gastrointestinal tract, specificallythe ileum, the rectum, the colon, the jejunum, and to a lesser extent inthe duodenum and stomach, suggests the TRP-PLIK2b polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing gastrointesinal diseases and/or disorders, which include, butare not limited to, ulcers, irritable bowel syndrome, inflammatory boweldisease, diarrhea, traveler's diarrhea, drug-related diarrhea polyps,absorption disorders, constipation, diverticulitis, vascular disease ofthe intestines, intestinal obstruction, intestinal infections,ulcerative colitis, Shigellosis, cholera, Crohn's Disease, amebiasis,enteric fever, Whipple's Disease, peritonitis, intrabdominal abcesses,hereditary hemochromatosis, gastroenteritis, viral gastroenteritis, foodpoisoning, mesenteric ischemia, mesenteric infarction, in addition to,metabolic diseases and/or disorders.

[0224] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosingsusceptibility to the following, non-limiting, gastrointestinalinfections: Salmonella infection, E. coli infection, E. coli 0157:H7infection, Shiga Toxin-producing E. coli infection, Campylobacterinfection (e.g., Campylobacter fetus, Campylobacter upsaliensis,Campylobacter hyointestinalis, Campylobacter lari, Campylobacter jejuni,Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum,Campylobacter rectus, Campylobacter curvus, Campylobacter sputorum,etc.), Heliobacter infection (e.g., Heliobacter cinaedi, Heliobacterfennelliae, etc.) Yersinia enterocolitica infection, Vibrio sp.Infection (e.g., Vibrio mimicus, Vibrio parahaemolyticus, Vibriofluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio vulnificus, Vibrioalginolyticus, Vibrio metschnikovii, Vibrio damsela, Vibriocincinnatiensis, etc.) Aeromonas infection (e.g., Aeromonas hydrophila,Aeromonas sobira, Aeromonas caviae, etc.), Plesiomonas shigelliodesinfection, Giardia infection (e.g., Giardia lamblia, etc.),Cryptosporidium infection, Listeria infection, Entamoeba histolyticainfection, Rotavirus infection, Norwalk virus infection, Clostridiumdifficile infection, Clostriudium perfringens infection, Staphylococcusinfection, Bacillus infection, in addition to any other gastrointestinaldisease and/or disorder implicated by the causative agents listed aboveor elsewhere herein.

[0225] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of the TRP-PLIK2polypeptide in kidney tissue suggests the TRP-PLIK2b polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome.for example.

[0226] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trpl2 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H.,Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0227] Thus, the TRP-PLIK2b polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein.

[0228] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of the TRP-PLIK2polypeptide in bone marrow tissue suggests the TRP-PLIK2bpolynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing immune diseases and/or disorders.Representative uses are described in the “Immune Activity”,“Chemotaxis”, and “Infectious Disease” sections below, and elsewhereherein. Briefly, the strong expression in immune tissue indicates a rolein regulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.

[0229] The TRP-PLIK2b polypeptide may also be useful as a preventativeagent for immunological disorders including arthritis, asthma,immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis,granulomatous disease, inflammatory bowel disease, sepsis, acne,neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cellmediated cytotoxicity; immune reactions to transplanted organs andtissues, such as host-versus-graft and graft-versus-host diseases, orautoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma. The TRP-PLIK2 polypeptide may be useful for modulatingcytokine production, antigen presentation, or other processes, such asfor boosting immune responses, etc.

[0230] Moreover, the protein may represent a factor that influences thedifferentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, astissuemarkers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0231] Significantly, TRP-PLIK2b is believed to represent the first TRPfamily member expressed in bone marrow tissue.

[0232] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of TRP-PLIK intestis tissue emphasizes the potential utility for TRP-PLIK2bpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0233] In preferred embodiments, TRP-PLIK2b polynucleotides andpolypeptides including agonists, antagonists, and/or fragments thereof,have uses which include treating, diagnosing, prognosing, and/orpreventing the following, non-limiting, diseases or disorders of thetestis: spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The TRP-PLIK2bpolynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0234] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for TRP-PLIK2b polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0235] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0236] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I.,Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(l):61-6, (1998)); Htrp-l (Zhu, X., Chu, P, B., Peyton, M.,Birnbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0237] Thus, the TRP-PLIK2b polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein.

[0238] The predominate differential expression of TRP-PLIK2 in prostatetumor relative to normal prostate tissue strongly suggests TRP-PLIK2bpolynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, have uses which include treating, diagnosing,prognosing, and/or preventing prostate cancers and/or proliferativeconditions.

[0239] Alternatively, the tissue distribution of TRP-PLIK2 in liverindicates the protein product of the TRP-PLIK2b clone would be usefulfor the detection and treatment of liver disorders and cancers.Representative uses are described in the “Hyperproliferative Disorders”,“Infectious Disease”, and “Binding Activity” sections below, andelsewhere herein. Briefly, the protein can be used for the detection,treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis,liver metabolic diseases and conditions that are attributable to thedifferentiation of hepatocyte progenitor cells, cirrhosis, hepaticcysts, pyrogenic abscess, amebic abcess, hydatid cyst,cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma,hepatocellulae carcinoma, cholangiocarcinoma, angiosarcoma, andgranulomatous liver disease.

[0240] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosing thefollowing, non-limiting, hepatic infections: liver disease caused bysepsis infection, liver disease caused by bacteremia, liver diseasecaused by Pneomococcal pneumonia infection, liver disease caused byToxic shock syndrome, liver disease caused by Listeriosis, liver diseasecaused by Legionnaries' disease, liver disease caused by Brucellosisinfection, liver disease caused by Neisseria gonorrhoeae infection,liver disease caused by Yersinia infection, liver disease caused bySalmonellosis, liver disease caused by Nocardiosis, liver disease causedby Spirochete infection, liver disease caused by Treponema palliduminfection, liver disease caused by Brrelia burgdorferi infection, liverdisease caused by Leptospirosis, liver disease caused by Coxiellaburnetii infection, liver disease caused by Rickettsia richettsiiinfection, liver disease caused by Chlamydia trachomatis infection,liver disease caused by Chlamydia psittaci infection, in addition to anyother hepatic disease and/or disorder implicated by the causative agentslisted above or elsewhere herein.

[0241] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0242] In preferred embodiments, TRP-PLIK2b polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0243] In more preferred embodiments, TRP-PLIK2b polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0244] TRP-PLIK2b polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since TRP-PLIK2 is dominantly expressed in bone marrow, the TRP-PLIK2bsplice variant may play an important role in regulating cytosolic C²⁺ inimmune system.

[0245] The TRP-PLIK2 gene maps to chromosome 9q21.2-22. 1. This regionis linked to amyotrophic lateral sclerosis with frontotemporal dementia,early-onset pulverulent cataract, infantile nephronophthisis,hypomagnesemia with secondary hypocalcemia and familial hemophagocyticlymphohistiocytosis. Therefore, agonists and/or antagonists of the novelTRP-PLIK2b splice variant can be used to treat diseases includingvarious forms of neuronal degeneration, neurogenic inflammation,allergy, immunodeficiency/excessive immune activation, visual defects,hearing disorder, pain, cancer, hypertension and other cardiovasculardiseases. In addition, the therapeutics may be useful in the treatmentof diseases associated with disturbances in Ca²⁺ homeostasis includingosteoporosis, hypercalciuric stone disease, and chronic renal failure.

[0246] In addition, TRP-PLIK2b polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ senstive proteins, the activation of Ca++ senstivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0247] The TRP-PLIK2b polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian bone marrow, kidney, testis, liver, small intestine, spinalcord, prostate, uterus, lung, lymph node, stomach, heart, brain, thymus,and pancreas, preferably human. TRP-PLIK2b polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, may be useful in diagnosing, treating, prognosing,and/or preventing immune, hematopoietic, renal, reproductive, hepatic,and/or proliferative diseases or disorders, particularly of the immunesystem.

[0248] In addition, antagonists of the TRP-PLIK2b polynucleotides andpolypeptides may have uses that include diagnosing, treating,prognosing, and/or preventing diseases or disorders related to transientreceptor potential channel activity, which may include immune,hematopoietic, renal, reproductive, hepatic, and/or proliferativediseases or disorders.

[0249] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those from CHAK1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the TRP-PLIK2bpolynucleotides, in addition to, other clones of the present invention,may be arrayed on microchips for expression profiling. Depending onwhich polynucleotide probe is used to hybridize to the slides, a changein expression of a specific gene may provide additional insight into thefunction of this gene based upon the conditions being studied. Forexample, an observed increase or decrease in expression levels when thepolynucleotide probe used comes from tissue that has been treated withknown immunoglobulin inhibitors, which include, but are not limited tothe drugs listed herein or otherwise known in the art, might indicate afunction in modulating immunoglobulin function, for example. In the caseof TRP-PLIK2b, bone marrow, kidney, testis, liver, small intestine,spinal cord, prostate, uterus, lung, lymph node, stomach, heart, brain,thymus, and/or pancrease, should be used to extract RNA to prepare theprobe.

[0250] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the TRP-PLIK2b gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:3 (FIGS. 2A-G).

[0251] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theTRP-PLIK2b, transforming yeast deficient in transient receptor potentialchannel activity with TRP-PLIK2b and assessing their ability to growwould provide convincing evidence the TRP-PLIK2b polypeptide hastransient receptor potential channel activity. Additional assayconditions and methods that may be used in assessing the function of thepolynucletides and polypeptides of the present invention are known inthe art, some of which are disclosed elsewhere herein.

[0252] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0253] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a bone marrow,kidney, testis, liver, small intestine, spinal cord, prostate, uterus,lung, lymph node, stomach, heart, brain, thymus, and/orpancrease-specific promoter), or it can be expressed at a specified timeof development using an inducible and/or a developmentally regulatedpromoter.

[0254] In the case of TRP-PLIK2b transgenic mice or rats, if nophenotype is apparent in normal growth conditions, observing theorganism under diseased conditions (immune, hematopoietic, renal,reproductive, hepatic, or proliferative disorders, etc.) may lead tounderstanding the function of the gene. Therefore, the application ofantisense and/or sense methodology to the creation of transgenic mice orrats to refine the biological function of the polypeptide is encompassedby the present invention.

[0255] In preferred embodiments, the following N-terminal TRP-PLIK2bdeletion polypeptides are encompassed by the present invention:M1-L1970, 12-L1970, 13-L1970, L4-L1970, S5-L1970, K6-L1970, S7-L1970,Q8-L1970, K9-L1970, S10-L1970, W11-L1970, 112-L1970, K13-L1970,G14-L1970, V15-L1970, F16-L1970, D17-L1970, K18-L1970, R19-L1970,E20-L1970, C21-L1970, S22-L1970, T23-L1970, 124-L1970, 125-L1970,P26-L1970, S27-L1970, S28-L1970, K29-L1970, N30-L1970, P31-L1970,H32-L1970, R33-L1970, C34-L1970, T35-L1970, P36-L1970, V37-L1970,C38-L1970, Q39-L1970, V40-L1970, C41-L1970, Q42-L1970, N43-L1970,L44-L1970, 145-L1970, R46-L1970, C47-L1970, Y48-L1970, C49-L1970,G50-L1970, R51-L1970, L52-L1970, 153-L1970, G54-L1970, D55-L1970,H56-L1970, A57-L1970, G58-L1970, 159-L1970, D60-L1970, Y61-L1970,S62-L1970, W63-L1970, T64-L1970, 165-L1970, S66-L1970, A67-L1970,A68-L1970, K69-L1970, G70-L1970, K71-L1970, E72-L1970, S73-L1970,E74-L1970, Q75-L1970, W76-L1970, S77-L1970, V78-L1970, E79-L1970,K80-L1970, H81-L1970, T82-L1970, T83-L1970, K84-L1970, S85-L1970,P86-L1970, T87-L1970, D88-L1970, T89-L1970, F90-L1970, G91-L1970,T92-L1970, 193-L1970, N94-L1970, F95-L1970, Q96-L1970, D97-L1970,G98-L1970, E99-L1970, H100-L1970, T101-L1970, H102-L1970, H103-L1970,A104-L1970, K105-L1970, Y106-L1970, 1107-L1970, R108-L1970, T109-L1970,SIIO-L1970, Y111-L1970, D112-L1970, T113-L1970, K114-L1970, L115-L1970,D116-L1970, H117-L1970, L118-L1970, L119-L1970, H120-L1970, L121-L1970,M122-L1970, L123-L1970, K124-L1970, E125-L1970, W126-L1970, K127-L1970,M128-L1970, E129-L1970, L130-L1970, P131-L1970, K132-L1970, L133-L1970,V134-L1970, 1135-L1970, S136-L1970, V137-L1970, H138-L1970, G139-L1970,G140-L1970, 1141-L1970, Q142-L1970, N143-L1970, F144-L1970, T145-L1970,M146-L1970, P147-L1970, S148-L1970, K149-L1970, F150-L1970, K151-L1970,E152-L1970, I153-L1970, F154-L1970, S155-L1970, Q156-L1970, G157-L1970,L158-L1970, V159-L1970, K160-L1970, A161-L1970, A162-L1970, E163-L1970,T164-L1970, T165-L1970, G166-L1970, A167-L1970, W168-L1970, I169-L1970,1170-L1970, T171-L1970, E172-L1970, G173-L1970, 1174-L1970, N175-L1970,T176-L1970, G177-L1970, V178-L1970, S179-L1970, K180-L1970, H181-L1970,V182-L1970, G183-L1970, D184-L1970, A185-L1970, L186-L1970, K187-L1970,S188-L1970, H189-L1970, S190-L1970, S191-L1970, H192-L1970, S193-L1970,L194-L1970, R195-L1970, K196-L1970, 1197-L1970, W198-L1970, T199-L1970,V200-L1970, G201-L1970, 1202-L1970, P203-L1970, P204-L1970, W205-L1970,G206-L1970, V207-L1970, 1208-L1970, E209-L1970, N210-L1970, Q211-L1970,R212-L1970, D213-L1970, L214-L1970, 1215-L1970, G216-L1970, K217-L1970,D218-L1970, V219-L1970, V220-L1970, C221-L1970, L222-L1970, Y223-L1970,Q224-L1970, T225-L1970, L226-L1970, D227-L1970, N228-L1970, P229-L1970,L230-L1970, S231-L1970, K232-L1970, L233-L1970, T234-L1970, T235-L1970,L236-L1970, N237-L1970, S238-L1970, M239-L1970, H240-L1970, S241-L1970,H242-L1970, F243-L1970, 1244-L1970, L245-L1970, S246-L1970, D247-L1970,D248-L1970, G249-L1970, T250-L1970, V251-L1970, G252-L1970, K253-L1970,Y254-L1970, G255-L1970, N256-L1970, E257-L1970, M258-L1970, K259-L1970,L260-L1970, R261-L1970, R262-L1970, N263-L1970, L264-L1970, E265-L1970,K266-L1970, Y267-L1970, L268-L1970, S269-L1970, L270-L1970, Q271-L1970,K272-L1970, 1273-L1970, H274-L1970, C275-L1970, R276-L1970, S277-L1970,R278-L1970, Q279-L1970, G280-L1970, V281-L1970, P282-L1970, V283-L1970,V284-L1970, G285-L1970, L286-L1970, V287-L1970, V288-L1970, E289-L1970,G290-L1970, G291-L1970, P292-LI 970, N293-L1970, V294-L1970, 1295-L1970,L296-L1970, S297-L1970, V298-L1970, W299-L1970, E300-L1970, T301-L1970,V302-L1970, K303-L1970, D304-L1970, K305-L1970, D306-L1970, P307-L1970,V308-L1970, V309-L1970, V310-L1970, C311-L1970, E312-L1970, G313-L1970,T314-L1970, G315-L1970, R316-L1970, A317-L1970, A318-L1970, D319-L1970,L320-L1970, L321-L1970, A322-L1970, F323-L1970, T324-L1970, H325-L1970,K326-L1970, H327-L1970, L328-L1970, A329-L1970, D330-L1970, E331-L1970,G332-L1970, M333-L1970, L334-L1970, R335-L1970, P336-L1970, Q337-L1970,V338-L1970, K339-L1970, E340-L1970, E341-L1970, 1342-L1970, 1343-L1970,C344-L1970, M345-L1970, 1346-L1970, Q347-L1970, N348-L1970, T349-L1970,F350-L1970, N351-L1970, F352-L1970, S353-L1970, L354-L1970, K355-L1970,Q356-L1970, S357-L1970, K358-L1970, H359-L1970, L360-L1970, F361-L1970,Q362-L1970, 1363-L1970, L364-L1970, M365-L1970, E366-L1970, C367-L1970,M368-L1970, V369-L1970, H370-L1970, R371-L1970, D372-L1970, C373-L1970,1374-L1970, T375-L1970, 1376-L1970, F377-L1970, D378-L1970, A379-L1970,D380-L1970, S381-L1970, E382-L1970, E383-L1970, Q384-L1970, Q385-L1970,D386-L1970, L387-L1970, D388-L1970, L389-L1970, A390-L1970, 1391-L1970,L392-L1970, T393-L1970, A394-L1970, L395-L1970, L396-L1970, K397-L1970,G398-L1970, T399-L1970, N400-L1970, L401-L1970, S402-L1970, A403-L1970,S404-L1970, E405-L1970, Q406-L1970, L407-L1970, N408-L1970, L409-L1970,A410-L1970, M411-L1970, A412-L1970, W413-L1970, D414-L1970, R415-L1970,V416-L1970, D417-L1970, 1418-L1970, A419-L1970, K420-L1970, K421-L1970,H422-L1970, 1423-L1970, L424-L1970, 1425-L1970, Y426-L1970, E427-L1970,Q428-L1970, H429-L1970, W430-L1970, K431-L1970, P432-L1970, D433-L1970,A434-L1970, L435-L1970, E436-L1970, Q437-L1970, A438-L1970, M439-L1970,S440-L1970, D441-L1970, A442-L1970, L443-L1970, V444-L1970, M445-L1970,D446-L1970, R447-L1970, V448-L1970, D449-L1970, F450-L1970, V451-L1970,K452-L1970, L453-L1970, L454-L1970, 1455-L1970, E456-L1970, Y457-L1970,G458-L1970, V459-L1970, N460-L1970, L461-L1970, H462-L1970, R463-L1970,F464-L1970, L465-L1970, T466-L1970, 1467-L1970, P468-L1970, R469-L1970,L470-L1970, E471-L1970, E472-L1970, L473-L1970, Y474-L1970, N475-L1970,T476-L1970, K477-L1970, Q478-L1970, G479-L1970, P480-L1970, T481-L1970,N482-L1970, T483-L1970, L484-L1970, L485-L1970, H486-L1970, H487-L1970,L488-L1970, V489-L1970, Q490-L1970, D491-L1970, V492-L1970, K493-L1970,Q494-L1970, and/or H495-L1970, of SEQ ID NO:4. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these N-terminal TRP-PLIK2b deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0256] In preferred embodiments, the following C-terminal TRP-PLIK2bdeletion polypeptides are encompassed by the present invention:M1-L1970, M1-Q1969, M1-M1968, M1-D1967, M1-D1966, M1-E1965, M1-P1964,M1-S1963, M1-N1962, M1-R1961, M1-G1960, M1-T1959, M1-E1958, M1-R1957,M1-A1956, M1-P1955, M1-P1954, M1-E1953, M1-E1952, M1-A1951, M1-S1950,M1-E1949, M1-I1948, M1-K1947, M1-I1946, M1-E1945, M1-L1944, M1-G1943,M1-F1942, M1-T1941, M1-S1940, M1-N1939, M1-I1938, M1-R1937, M1-E1936,M1-P1935, M1-S1934, M1-Y1933, M1-D1932, M1-N1931, M1-R1930, M1-K1929,M1-L1928, M1-D1927, M1-P1926, M1-L1925, M1-K1924, M1-L1923, M1-K1922,M1-R1921, M1-C1920, M1-C1919, M1-S1918, M1-N1917, M1-C1916, M1-H1915,M1-H1914, M1-K1913, M1-A1912, M1-I1911, M1-F1910, M1-N1909, M1-R1908,M1-I1907, M1-A1906, M1-D1905, M1-E1904, M1-G1903, M1-L1902, M1-N1901,M1-A1900, M1-P1899, M1-G1898, M1-F1897, M1-V1896, M1-M1895, M1-G1894,M1-R1893, M1-S1892, M1-Q1891, M1-K1890, M1-V1889, M1-E1888, M1-P1887,M1-K1886, M1-I1885, M1-V1884, M1-S1883, M1-P1882, M1-D1881, M1-T1880,M1-L1879, M1-N1878, M1-E1877, M1-G1876, M1-V1875, M1-G1874, M1-Q1873,M1-L1872, M1-D1871, M1-L1870, M1-V1869, M1-L1868, M1-L1867, M1-E1866,M1-G1865, M1-R1864, M1-T1863, M1-Y1862, M1-E1861, M1-Y1860, M1-T1859,M1-W1858, M1-H1857, M1-S1856, M1-F1855, M1-A1854, M1-L1853, M1-M1852,M1-L1851, M1-E1850, M1-E1849, M1-L1848, M1-T1847, M1-N1846, M1-T1845,M1-P1844, M1-T1843, M1-I1842, M1-E1841, M1-D1840, M1-G1839, M1-N1838,M1-N1837, M1-N1836, M1-N1835, M1-Y1834, M1-K1833, M1-R1832, M1-F1831,M1-E1830, M1-G1829, M1-T1828, M1-M1827, M1-Y1826, M1-K1825, M1-E1824,M1-I1823, M1-T1822, M1-L1821, M1-W1820, M1-Q1819, M1-N1818, M1-A1817,M1-S1816, M1-H1815, M1-C1814, M1-Y1813, M1-I1812, M1-L1811, M1-F1810,M1-V1809, M1-E1808, M1-L1807, M1-F1806, M1-R1805, M1-P1804, M1-T1803,M1-Y1802, M1-P1801, M1-I1800, M1-T1799, M1-Q1798, M1-P1797, M1-K1796,M1-V1795, M1-Q1794, M1-N1793, M1-F1792, M1-T1791, M1-Y1790, M1-I1789,M1-L1788, M1-K1787, M1-Q1786, M1-A1785, M1-A1784, M1-R1783, M1-Q1782,M1-Q1781, M1-Q1780, M1-I1779, M1-E1778, M1-R1777, M1-L1776, M1-C1775,M1-L1774, M1-H1773, M1-L1772, M1-V1771, M1-T1770, M1-S1769, M1-E1768,M1-Q1767, M1-F1766, M1-I1765, M1-K1764, M1-H1763, M1-W1762, M1-T1761,M1-R1760, M1-V1759, M1-V1758, M1-E1757, M1-P1756, M1-L1755, M1-F1754,M1-S1753, M1-K1752, M1-V1751, M1-I1750, M1-F1749, M1-V1748, M1-Q1747,M1-G1746, M1-P1745, M1-K1744, M1-L1743, M1-I1742, M1-D1741, M1-D1740,M1-E1739, M1-S1738, M1-W1737, M1-T1736, M1-S1735, M1-V1734, M1-V1733,M1-R1732, M1-M1731, M1-A1730, M1-K1729, M1-R1728, M1-L1727, M1-G1726,M1-G1725, M1-D1724, M1-M1723, M1-E1722, M1-E1721, M1-R1720, M1-S1719,M1-L1718, M1-V1717, M1-Q1716, M1-I1715, M1-M1714, M1-A1713, M1-A1712,M1-R1711, M1-G1710, M1-R1709, M1-Q1708, M1-S1707, M1-W1706, M1-S1705,M1-S1704, M1-M1703, M1-S1702, M1-K1701, M1-D1700, M1-L1699, M1-N1698,M1-L1697, M1-P1696, M1-S1695, M1-S1694, M1-E1693, M1-E1692, M1-L1691,M1-R1690, M1-Y1689, M1-V1688, M1-T1687, M1-I1686, M1-E1685, M1-E1684,M1-G1683, M1-A1682, M1-F1681, M1-L1680, M1-Q1679, M1-V1678, M1-P1677,M1-T1676, M1-F1675, M1-P1674, M1-I1673, M1-T1672, M1-Q1671, M1-S1670,M1-L1669, M1-R1668, M1-M1667, M1-L1666, M1-N1665, M1-N1664, M1-R1663,M1-E1662, M1-I1661, M1-A1660, M1-S1659, M1-Y1658, M1-H1657, M1-H1656,M1-H1655, M1-P1654, M1-E1653, M1-Q1652, M1-P1651, M1-S1650, M1-K1649,M1-L1648, M1-S1647, M1-A1646, M1-S1645, M1-I1644, M1-K1643, M1-D1642,M1-V1641, M1-G1640, M1-I1639, M1-S1638, M1-S1637, M1-K1636, M1-L1635,M1-L1634, M1-S1633, M1-N1632, M1-R1631, M1-N1630, M1-L1629, M1-N1628,M1-T1627, M1-S1626, M1-R1625, M1-S1624, M1-N1623, M1-W1622, M1-L1621,M1-S1620, M1-N1619, M1-K1618, M1-S1617, M1-L1616, M1-D1615, M1-E1614,M1-Q1613, M1-S1612, M1-Q1611, M1-K1610, M1-L1609, M1-Y1608, M1-D1607,M1-S1606, M1-I1605, M1-Q1604, M1-I1603, M1-A1602, M1-C1601, M1-Q1600,M1-G1599, M1-I1598, M1-E1597, M1-K1596, M1-T1595, M1-K1594, M1-M1593,M1-K1592, M1-Q1591, M1-H1590, M1-I1589, M1-Y1588, M1-P1587, M1-E1586,M1-V1585, M1-G1584, M1-T1583, M1-H1582, M1-S1581, M1-F1580, M1-K1579,M1-S1578, M1-V1577, M1-T1576, M1-F1575, M1-W1574, M1-N1573, M1-K1572,M1-S1571, M1-Y1570, M1-E1569, M1-E1568, M1-E1567, M1-S1566, M1-I1565,M1-S1564, M1-N1563, M1-E1562, M1-G1561, M1-P1560, M1-E1559, M1-P1558,M1-N1557, M1-L1556, M1-Q1555, M1-D1554, M1-S1553, M1-Q1552, M1-S1551,M1-C1550, M1-A1549, M1-N1548, M1-V1547, M1-T1546, M1-I1545, M1-I1544,M1-P1543, M1-V1542, M1-Q1541, M1-L1540, M1-G1539, M1-Q1538, M1-T1537,M1-N1536, M1-K1535, M1-K1534, M1-K1533, M1-K1532, M1-S1531, M1-L1530,M1-R1529, M1-R1528, M1-D1527, M1-K1526, M1-T1525, M1-L1524, M1-M1523,M1-K1522, M1-A1521, M1-K1520, M1-V1519, M1-W1518, M1-A1517, M1-G1516,M1-Q1515, M1-G1514, M1-I1513, M1-E1512, M1-S1511, M1-S1510, M1-G1509,M1-S1508, M1-L1507, M1-N1506, M1-K1505, M1-I1504, M1-K1503, M1-C1502,M1-I1501, M1-K1500, M1-M1499, M1-L1498, M1-K1497, M1-E1496, M1-E1495,M1-K1494, M1-H1493, M1-F1492, M1-R1491, M1-F1490, M1-S1489, M1-H1488,M1-S1487, M1-R1486, M1-A1485, M1-Fl484, M1-P1483, M1-R1482, M1-Y1481,M1-R1480, M1-R1479, M1-L1478, M1-P1477, M1-N1476, M1-I1475, M1-W1474,M1-F1473, M1-S1472, M1-T1471, M1-N1470, M1-P1469, M1-Q1468, M1-L1467,M1-W1466, M1-P1465, M1-G1464, M1-V1463, M1-E1462, M1-S1461, M1-C1460,M1-E1459, M1-S1458, M1-S1457, M1-Q1456, M1-A1455, M1-S1454, M1-R1453,M1-T1452, M1-S1451, M1-N1450, M1-D1449, M1-S1448, M1-L1447, M1-S1446,M1-S1445, M1-D1444, M1-Q1443, M1-A1442, M1-Q1441, M1-K1440, M1-Q1439,M1-H1438, M1-Q1437, M1-E1436, M1-S1435, M1-R1434, M1-S1433, M1-S1432,M1-D1431, M1-S1430, M1-D1429, M1-C1428, M1-T1427, M1-S1426, M1-P1425,M1-L1424, M1-C1423, M1-T1422, M1-Q1421, M1-W1420, M1-K1419, M1-K1418,M1-K1417, M1-I1416, M1-S1415, M1-F1414, M1-V1413, M1-G1412, M1-T1411,M1-E1410, M1-D1409, M1-G1408, M1-E1407, M1-S1406, M1-F1405, M1-A1404,M1-W1403, M1-N1402, M1-V1401, M1-Y1400, M1-G1399, M1-G1398, M1-G1397,M1-T1396, M1-Q1395, M1-M1394, M1-I1393, M1-K1392, M1-A1391, M1-Q1390,M1-S1389, M1-L1388, M1-P1387, M1-S1386, M1-S1385, M1-M1384, M1-T1383,M1-M1382, M1-P1381, M1-E1380, M1-P1379, M1-T1378, M1-C1377, M1-S1376,M1-L1375, M1-T1374, M1-P1373, M1-L1372, M1-V1371, M1-Q1370, M1-E1369,M1-A1368, M1-K1367, M1-D1366, M1-Q1365, M1-G1364, M1-D1363, M1-L1362,M1-L1361, M1-H1360, M1-A1359, M1-I1358, M1-P1357, M1-E1356, M1-H1355,M1-K1354, M1-E1353, M1-K1352, M1-P1351, M1-E1350, M1-D1349, M1-V1348,M1-S1347, M1-A1346, M1-W1345, M1-D1344, M1-S1343, M1-V1342, M1-V1341,M1-P1340, M1-T1339, M1-Q1338, M1-G1337, M1-T1336, M1-L1335, M1-H1334,M1-V1333, M1-L1332, M1-V1331, M1-E1330, M1-T1329, M1-Q1328, M1-I1327,M1-D1326, M1-Q1325, M1-E1324, M1-T1323, M1-A1322, M1-L1321, M1-V1320,M1-D1319, M1-P1318, M1-V1317, M1-S1316, M1-P1315, M1-R1314, M1-S1313,M1-L1312, M1-P1311, M1-L1310, M1-V1309, M1-T1308, M1-E1307, M1-A1306,M1-S1305, M1-F1304, M1-P1303, M1-V1302, M1-R1301, M1-K1300, M1-L1299,M1-N1298, M1-S1297, M1-P1296, M1-V1295, M1-L1294, M1-L1293, M1-F1292,M1-Q1291, M1-G1290, M1-Y1289, M1-K1288, M1-S1287, M1-H1286, M1-A1285,M1-Q1284, M1-R1283, M1-N1282, M1-P1281, M1-S1280, M1-V1279, M1-G1278,M1-S1277, M1-V1276, M1-V1275, M1-I1274, M1-S1273, M1-S1272, M1-Q1271,M1-T1270, M1-E1269, M1-Q1268, M1-R1267, M1-E1266, M1-Q1265, M1-D1264,M1-N1263, M1-R1262, M1-V1261, M1-N1260, M1-T1259, M1-A1258, M1-E1257,M1-R1256, M1-K1255, M1-S1254, M1-N1253, M1-T1252, M1-I1251, M1-E1250,M1-L1249, M1-L1248, M1-A1247, M1-G1246, M1-R1245, M1-Q1244, M1-V1243,M1-R1242, M1-P1241, M1-P1240, M1-H1239, M1-R1238, M1-G1237, M1-G1236,M1-A1235, M1-L1234, M1-S1233, M1-R1232, M1-L1231, M1-L1230, M1-S1229,M1-S1228, M1-P1227, M1-M1226, M1-S1225, M1-Y1224, M1-Y1223, M1-Q1222,M1-Y1221, M1-K1220, M1-K1219, M1-E1218, M1-G1217, M1-A1216, M1-I1215,M1-E1214, M1-M1213, M1-S1212, M1-G1211, M1-L1210, M1-V1209, M1-E1208,M1-A1207, M1-C1206, M1-I1205, M1-V1204, M1-N1203, M1-S1202, M1-W1201,M1-S1200, M1-H1199, M1-P1198, M1-L1197, M1-K1196, M1-Ki195, M1-C1194,M1-T1193, M1-S1192, M1-H1191, M1-K1190, M1-R1189, M1-K1188, M1-A1187,M1-L1186, M1-L1185, M1-A1184, M1-E1183, M1-D1182, M1-E1181, M1-Q1180,M1-L1179, M1-T1178, M1-D1177, M1-V1176, M1-A1175, M1-S1174, M1-L1173,M1-V1172, M1-K1171, M1-L1170, M1-T1169, M1-D1168, M1-V1167, M1-T1166,M1-L1165, M1-A1164, M1-S1163, M1-L1162, M1-D1161, M1-Q1160, M1-L1159,M1-H1158, M1-G1157, M1-V1156, M1-Q1155, M1-S1154, M1-DI153, M1-L1152,M1-S1151, M1-L1150, M1-L1149, M1-S1148, M1-DI147, M1-K1146, M1-I1145,M1-F1144, M1-S1143, M1-V1142, M1-K1141, M1-E1140, M1-N1139, M1-M1138,M1-E1137, M1-K1136, M1-L1135, M1-Q1134, M1-F1133, M1-Y1132, M1-M1131,M1-E1130, M1-T1129, M1-V1128, M1-R1127, M1-E1126, M1-S1125, M1-T1124,M1-V1123, M1-R1122, M1-I1121, M1-R1120, M1-E1119, M1-E1118, M1-C1117,M1-S1116, M1-C1115, M1-N1114, M1-V1113, M1-D1112, M1-E1111, M1-M1110,M1-K1109, M1-E1108, M1-H1107, M1-F1106, M1-Y1105, M1-K1104, M1-E1103,M1-V1102, M1-C1101, M1-Q1100, M1-E1099, M1-E1098, M1-F1097, M1-D1096,M1-H1095, M1-L1094, M1-K1093, M1-K1092, M1-L1091, M1-DIO90, M1-E1089,M1-K1088, M1-S1087, M1-L1086, M1-Y1085, M1-L1084, M1-K1083, M1-L1082,M1-G1081, M1-V1080, M1-D1079, M1-G1078, M1-E1077, M1-E1076, M1-Q1075,M1-D1074, M1-H1073, M1-P1072, M1-A1071, M1-R1070, M1-H1069, M1-C1068,M1-C1067, M1-L1066, M1-R1065, M1-R1064, M1-L1063, M1-L1062, M1-L1061,M1-GiO60, M1-V1059, M1-H1058, M1-S1057, M1-L1056, M1-L1055, M1-11054,M1-L1053, M1-P1052, M1-P1051, M1-P1050, M1-L1049, M1-W1048, M1-P1047,M1-K1046, M1-E1045, M1-H1044, M1-Y1043, M1-T1042, M1-M1041, M1-I1040,M1-Y1039, M1-R1038, M1-Y1037, M1-R1036, M1-N1035, M1-Y1034, M1-K1033,M1-W1032, M1-L1031, M1-N1030, M1-N1029, M1-S1028, M1-I1027, M1-S1026,M1-E1025, M1-M1024, M1-D1023, M1-L1022, M1-Y1021, M1-V1020, M1-N1019,M1-N1018, M1-F1017, M1-F1016, M1-A1015, M1-I1014, M1-L1013, M1-L1012,M1-N1011, M1-V1010, M1-M1009, M1-I1008, M1-I1007, M1-Y1006, M1-Q1005,M1-V1004, M1-F1003, M1-L1002, M1-Y1001, M1-V1000, M1-A999, M1-Q998,M1-L997, M1-F996, M1-P995, M1-T994, M1-L993, M1-F992, M1-S991, M1-G990,M1-P989, M1-P988, M1-C987, M1-S986, M1-P985, M1-Q984, M1-S983, M1-S982,M1-C981, M1-V980, M1-D979, M1-1978, M1-E977, M1-G976, M1-A975, M1-Y974,M1-V973, M1-E972, M1-G971, M1-Y970, M1-1969, M1-M968, M1-W967, M1-Y966,M1-P965, M1-E964, M1-F963, M1-V962, M1-1961, M I-D960, M1-R959, M1-A958,M1-L957, M1-S956, M1-W955, M1-S954, M1-P953, M1-P952, M1-E951, M1-K950,M1-P949, M1-S948, M1-L947, M1-1946, M1-A945, M1-K944, Ml -R943, M1-A942,M1-V941, M1-G940, M1-F939, M1-S938, M1-L937, M1-L936, M1-V935, M1-1934,M1-A933, M1-M932, M1-1931, M1-1930, M1-V929, M1-1928, M1-Y927, M1-F926,M1-M925, M1-N924, M1-A923, M1-T922, M1-M921, M1-K920, M1-A919, M1-1918,M1-M917, M1-T916, M1-V915, M1-Y914, M1-P913, M1-G912, M1-A911, M1-H910,M1-Q909, M1-N908, M1-V907, M1-A906, M1-F905, M1-F904, M1-D903, M1-L902,M1-L901, M1-R900, M1-S899, M1-F898, M1-W897, M1-F896, M1-1895, M1-1894,M1-D893, M1-1892, M1-C891, M1-Y890, M1-1889, M1-L888, M1-R887, M1-G886,M1-A885, M1-T884, M1-H883, M1-F882, M1-P881, M1-P880, M1-D879, M1-G878,M1-W877, M1-R876, M1-L875, M1-V874, M1-F873, M1-G872, M1-A871, M1-S870,M1-F869, M1-L868, M1-G867, M1-1866, M1-A865, M1-V864, M1-T863, M1-E862,M1-T861, M1-L860, M1-N859, M1-W858, M1-Y857, M1-E856, M1-S855, M1-1854,M1-W853, M1-V852, M1-K851, M1-V850, M1-K849, M1-Q848, M1-T847, M1-F846,M1-K845, M1-G844, M1-P843, M1-E842, M1-S841, M1-1840, M1-C839, M1-1838,M1-E837, M1-R836, M1-V835, M1-V834, M1-E833, M1-1832, M1-A831, M1-N830,M1-T829, M1-F828, M1-1827, M1-Y826, M1-1825, M1-S824, M1-V823, M1-L822,M1-W821, M1-E820, M1-Q819, M1-V818, M1-S817, M1-P816, M1-Q815, M1-P814,M1-Q813, M1-M812, M1-E811, M1-V810, M1-L809, M1-V808, M1-T807, M1-Y806,M1-T805, M1-F804, M1-L803, M1-M802, M1-L801, M1-F800, M1-A799, M1-L798,M1-Y797, M1-A796, M1-M795, M1-T794, M1-Y793, M1-F792, M1-W791, M1-F790,M1-K789, M1-V788, M1-1787, M1-P786, M1-A785, M1-S784, M1-Y783, M1-F782,M1-E781, M1-Y780, M1-V779, M1-K778, M1-R777, M1-T776, M1-W775, M1-P774,M1-L773, M1-H772, M1-Q771, M1-H770, M1-G769, M1-S768, M1-E767, M1-L766,M1-G765, M1-F764, M1-H763, M1-Q762, M1-N761, M1-E760, M1-D759, M1-L758,M1-K757, M1-E756, M1-D755, M1-H754, M1-G753, M1-R752, M1-E751, M1-L750,M1-D749, M1-Y748, M1-E747, M1-K746, M1-V745, M1-S744, M1-A743, M1-S742,M1-E741, M1-K740, M1-S739, M1-S738, M1-S737, M1-A736, M1-N735, M1-Q734,M1-D733, M1-S732, M1-Y731, M1-Y730, M1-W729, M1-M728, M1-F727, M1-Q726,M1-F725, M1-D724, M1-Q723, M1-S722, M1-Q721, M1-P720, M1-V719, M1-H718,M1-S717, M1-M716, M1-E715, M1-A714, M1-K713, M1-S712, M1-K711, M1-F710,M1-E709, M1-L708, M1-T707, M1-L706, M1-1705, M1-T704, M1-P703, M1-P702,M1-L701, M1-1700, M1-1699, M1-S698, M1-1697, M1-I696, M1-1695, M1-K694,M1-L693, M1-W692, M1-S691, M1-N690, M1-K689, M1-R688, M1-M687, M1-K686,M1-L685, M1-R684, M1-G683, M1-M682, M1-W681, M1-M680, M1-D679, M1-T678,M1-L677, M1-L676, M1-M675, M1-Q674, M1-T673, M1-C672, M1-T671, M1-H670,M1-S669, M1-V668, M1-F667, M1-P666, M1-R665, M1-L664, M1-G663, M1-G662,M1-S661, M1-V660, M1-A659, M1-L658, M1-K657, M1-L656, M1-C655, M1-T654,M1-S653, M1-N652, M1-S651, M1-W650, M1-N649, M1-R648, M1-L647, M1-E646,M1-Y645, M1-T644, M1-L643, M1-L642, M1-T641, M1-M640, M1-A639, M1-M638,M1-R637, M1-E636, M1-N635, M1-Q634, M1-K633, M1-F632, M1-A631, M1-K630,M1-E629, M1-L628, M1-L627, M1-D626, M1-L625, M1-A624, M1-L623, M1-Q622,M1-G621, M1-F620, M1-Q619, M1-K618, M1-S617, M1-Y616, M1-N615, M1-K614,M1-L613, M1-E612, M1-E611, M1-S610, M1-A609, M1-D608, M1-D607, M1-V606,M1-M605, M1-H604, M1-S603, M1-E602, M1-K601, M1-A600, M1-E599, M1-H598,M1-A597, M1-M596, M1-A595, M1-R594, M1-Y593, M1-L592, M1-1591, M1-C590,M1-A589, M1-1588, M1-V587, M1-A586, M1-K585, M1-V584, M1-T583, M1-A582,M1-E581, M1-E580, M1-G579, M1-H578, M1-Q577, M1-W576, M1-F575, M1-F574,M1-M573, M1-A572, M1-M571, M1-K570, M1-Q569, M1-R568, M1-K567, M1-M566,M1-L565, M1-V564, M1-A563, M1-W562, M1-V561, M1-L560, M1-L559, M1-D558,M1-N557, M1-Y556, M1-P555, M1-Y554, M1-L553, M1-F552, M1-G551, M1-T550,M1-S549, M1-E548, M1-P547, M1-D546, M1-D545, M1-S544, M1-V543, M1-N542,M1-Q541, M1-E540, M1-K539, M1-S538, M1-K537, M1-K536, M1-R535, M1-S534,M1-K533, M1-H532, M1-L531, M1-V530, M1-1529, M1-S528, M1-K527, M1-E526,M1-K525, M1-F524, M1-K523, M1-Y522, M1-P521, M1-Q520, M1-A519, M1-T518,M1-R517, M1-1516, M1-F515, M1-Q514, M1-S513, M1-H512, M1-L511, M1-T510,M1-S509, M1-E508, M1-A507, M1-S506, M1-E505, M1-N504, M1-R503, M1-N502,M1-G501, M1-S500, M1-S499, M1-H498, M1-R497, and/or M1-Q496 of SEQ IDNO:4. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseC-terminal TRP-PLIK2b deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0257] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the TRP-PLIK2b polypeptide (e.g., any combination ofboth N- and C- terminal TRP-PLIK2b polypeptide deletions) of SEQ IDNO:4. For example, internal regions could be defined by the equation:amino acid NX to amino acid CX, wherein NX refers to any N-terminaldeletion polypeptide amino acid of TRP-PLIK2b (SEQ ID NO:4), and whereCX refers to any C-terminal deletion polypeptide amino acid ofTRP-PLIK2b (SEQ ID NO:4). Polynucleotides encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese polypeptides as an immunogenic and/or antigenic epitope asdescribed elsewhere herein.

[0258] The TRP-PLIK2b polypeptides of the present invention weredetermined to comprise several phosphorylation sites based upon theMotif algorithm (Genetics Computer Group, Inc.). The phosphorylation ofsuch sites may regulate some biological activity of the TRP-PLIK2bpolypeptide. For example, phosphorylation at specific sites may beinvolved in regulating the proteins ability to associate or bind toother molecules (e.g., proteins, ligands, substrates, DNA, etc.). In thepresent case, phosphorylation may modulate the ability of the TRP-PLIK2bpolypeptide to associate with other potassium channel alpha subunits,beta subunits, or its ability to modulate potassium channel function.

[0259] The TRP-PLIK2b polypeptide was predicted to comprise twenty eightPKC phosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. . . .260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0260] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: IILSKSQKSWIKG(SEQ ID NO:115), STIIPSSKNPHRC (SEQ ID NO:116), SVEKHTTKSPTDT (SEQ IDNO:117), SHSSHSLRKIWTV (SEQ ID NO:118), LSVWETVKDKDPV (SEQ ID NO:119),VVCEGTGRAADLL (SEQ ID NO:120), DLLAFTHKHLADE (SEQ ID NO:121),NTFNFSLKQSKHL (SEQ ID NO:122), IVLHKSRKKSKEQ (SEQ ID NO:123),HGEEATVKAVIAC (SEQ ID NO:124), DQNASSSKESASV (SEQ ID NO:125),SKESASVKEYDLE (SEQ ID NO:126), QHLPWTRKVYEFY (SEQ ID NO:127),EPGKFTQKVKVWI (SEQ ID NO:128), RKAILSPKEPPSW (SEQ ID NO:129),RIRVTSERVTEMY (SEQ ID NO:130), ALTVDTLKVLSAV (SEQ ID NO:131),KRKHSTCKKLPHS (SEQ ID NO:132), LEITNSKREATNV (SEQ ID NO:133),ETGVFSIKKKWQT (SEQ ID NO:134), TCDSDSSRSEQHQ (SEQ ID NO:135),SLSDNSTRSAQSS (SEQ ID NO:136), FARSHSFRFHKEE (SEQ ID NO:137),KDRRLSKKKKNTQ (SEQ ID NO:138), DKISASLKSPQEP (SEQ ID NO:139),SMSSWSQRGRAAM (SEQ ID NO:140), QTIPYTPRFLEVF (SEQ ID NO:141), and/orPPARETGRNSPED (SEQ ID NO:142). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these TRP-PLIK2b PKC phosphorylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0261] The present invention also encompasses immunogenic and/orantigenic epitopes of the TRP-PLIK2b polypeptide.

[0262] The TRP-PLIK2b polypeptide has been shown to comprise sixteenglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0263] Asparagine phosphorylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. . .. 265:11397-I1404(1990).

[0264] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:HGGIQNFTMPSKFK (SEQ ID NO:143), IQNTFNFSLKQSKH (SEQ ID NO:144),LLKGTNLSASEQLN (SEQ ID NO:145), SSGNRNESAESTLH (SEQ ID NO:146),KSKEQNVSDDPEST (SEQ ID NO:147), SEELKNYSKQFGQL (SEQ ID NO:148),TYELRNWSNSTCLK (SEQ ID NO:149), LRNWSNSTCLKLAV (SEQ ID NO:150),YYSDQNASSSKESA (SEQ ID NO:151), ISEYWNLTETVAIG (SEQ ID NO:152),KMEDVNCSCEERIR (SEQ ID NO 153), SSLSDNSTRSAQSS (SEQ ID NO:154),PWLQPNTSFWINPL (SEQ ID NO:155), ICKIKNLSGSSEIG (SEQ ID NO:156),QGVGENLTDPSVIK (SEQ ID NO:157), and/or SPERINSTFGLEIK (SEQ ID NO:158).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these TRP-PLIK2basparagine glycosylation site polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0265] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:3 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5899 ofSEQ ID NO:3, b is an integer between 15 to 5913, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:3,and where b is greater than or equal to a+14.

[0266] Features of the Polypeptide Encoded by Gene No:3

[0267] The polypeptide of this gene provided as SEQ ID NO:6 (FIGS.3A-G), encoded by the polynucleotide sequence according to SEQ ID NO:5(FIGS. 3A-G), and/or encoded by the polynucleotide contained within thedeposited clone, TRP-PLIK2c, has significant homology at the nucleotideand amino acid level to the human channel-kinase 1 protein, also knownas the human CHAK1 or TRP-PLIKB1 protein (CHAK1; Genbank Accession No.gilAF346629; SEQ ID NO:9); and the human melastatin 1 protein(Melastatinl; Genbank Accession No. gil3243075; SEQ ID NO:260). Analignment of the TRP-PLIK2c polypeptide with this protein is provided inFIGS. 5A-F.

[0268] The TRP-PLIK2c polypeptide was determined to share 58.5% identityand 66.5% similarity with the human CHAK1 or TRP-PLIKBI protein (CHAK1;Genbank Accession No. gilAF346629; SEQ ID NO:9); and was determined toshare 47.8% identity and 58.7% similarity with the human melastatin 1protein (Melastatinl; Genbank Accession No. gil3243075; SEQ ID NO:260)as shown in FIG. 9.

[0269] The CHAK1 protein is believed to represent a member of a newclass of protein kianses referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain.

[0270] The melastatin 1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin 1 could represent a noveltherapeutic in the treatment of melanoa and potentially other cancers.

[0271] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the CHAK1 and melastatin 1 proteins, in addition to,other transient receptor potential channel family members referencedelsewhere herein or otherwise known in the art.

[0272] The TRP-PLIK2c (SEQ ID NO:6) polypeptide represents a novelsplice variant form of the TRP-PLIK2 (SEQ ID NO:2) polypeptide of thepresent invention.

[0273] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theTRP-PLIK2c polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 3A-G. The transmembrane domains arelocated from about amino acid 662 to about amino acid 679 (TM1), fromabout amino acid 756 to about amino acid 773 (TM2), from about aminoacid 830 to about amino acid 842 (TM3), from about amino acid 856 toabout amino acid 873 (TM4), from about amino acid 890 to about aminoacid 907 (TM5), and/or from about amino acid 965 to about amino acid 984(TM6) of SEQ ID NO:6. In this context, the term “about” may be construedto mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond theN-Terminus and/or C-terminus of the above referenced polypeptide.

[0274] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKIIIS1ILPPTILTLEF (SEQ ID NO:159), IVKFWFYTMAYLAFLMLF (SEQ ID NO:160),TETVAIGLFSAGF (SEQ ID NO:161), RLIYCIDIFWFSRLLDF (SEQ ID NO:162),MTANMFYIVIIMAIVLLS (SEQ ID NO:163), and/or FLQAVYLFVQYIIMVNLLIA (SEQ IDNO:164). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the TRP-PLIK2ctransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0275] In preferred embodiments, the present invention encompasses theuse of N-terminal deletions, C-terminal deletions, or any combination ofN-terminal and C-terminal deletions of any one or more of the TRP-PLIK2cTM1 thru TM6 transmembrane domain polypeptides as antigenic and/orimmunogenic epitopes.

[0276] In preferred embodiments, the present invention also encompassesthe use of N-terminal deletions, C-terminal deletions, or anycombination of N-terminal and C-terminal deletions of any one or more ofthe amino acids intervening (i.e., ion channel extracellular orintracellular loops) the TRP-PLIK2c TM1 thru TM6 transmembrane domainpolypeptides as antigenic and/or immunogenic epitopes.

[0277] The TRP-PLIK2c polypeptide was determined to comprise severalconserved cysteines, at amino acid 21, 34, 38, 41, 47, 49, 311, 367,590, 655, 672, 891, 981, 987, 1067, 1101, 1775, 1814, 1915, 1919, and1920 of SEQ ID No: 6 (FIGS. 3A-G). Conservation of cysteines at keyamino acid residues is indicative of conserved structural features,which may correlate with conservation of protein function and/oractivity.

[0278] In confirmation of the TRP-PLIK2c representing a member of thetransient receptor channel family, the TRP-PLIK2c polypeptide wasdetermined to comprise a predicted TRP domain (LWKYNR) located fromabout amino acid 1000 to about amino acid 1005 of SEQ ID NO:6. In thiscontext, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of theabove referenced polypeptide.

[0279] In further confirmation of the TRP-PLIK2c representing a memberof the transient receptor channel family, the TRP-PLIK2c polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 826 to about amino acid 986 of SEQ ID NO:6.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0280] The TRP-PLIK2c polypeptide was determined to comprise a predictednucleotide binding domain located from about amino acid 1867 to aboutamino acid 1872 of SEQ ID NO:6. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0281] In addition, the TRP-PLIK2c polypeptide was determined tocomprise a predicted zinc finger domain located at about amino acid 1882to about amino acid 1892 of SEQ ID NO:6. In this context, the term“about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids beyond the N-Terminus and/or C-terminus of the above referencedpolypeptide.

[0282] TRP-PLIK2c polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofTRP-PLIK2c by identifying mutations in the TRP-PLIK2c gene usingTRP-PLIK2c sequences as probes or by determining TRP-PLIK2c protein ormRNA expression levels. TRP-PLIK2c polypeptides will be useful inscreens for compounds that affect the activity of the protein.TRP-PLIK2c peptides can also be used for the generation of specificantibodies and as bait in yeast two hybrid screens to find proteins thespecifically interact with TRP-PLIK2c.

[0283] Expression profiling designed to measure the steady state mRNAlevels encoding the TRP-PLIK2 polypeptide showed predominately highexpression levels in bone marrow, kidney, and testis. The TRP-PLIK2polypeptide was also significantly expressioned in liver, and to alesser extent, in small intestine, spinal cord, prostate, uterus, lung,lymph node, stomach, heart, brain, thymus, and pancrease (as shown inFIG. 7). The tissue expression of TRP-PLIK2c may follow the same patternas for the TRP-PLIK2 polypeptide of the present invention.

[0284] Expanded analysis of TRP-PLIK2 expression levels by TaqMan™quantitative PCR (see FIG. 12) confirmed that the TRP-PLIK2 polypeptideis expressed in kidney, colon, and testis (FIG. 7). TRP-PLIK2 mRNA wasexpressed predominately in the lower gastrointestinal tract,specifically the ileum, the rectum, the colon, the jejunum, and to alesser extent in the duodenum and stomach. Significant expression wasobserved in the kidney, particularly in the cortex, followed by themedulla, and to a lesser extent in the testis, pelvis, and bone marrow(mononuclear cells).

[0285] Furthermore, an expanded analysis of TRP-PLIK2 expression levelsin various tumor and normal tissues by TaqMan™ quantitative PCR (seeFIG. 14) showed TRP-PLIK2 mRNA was differentially expressed to thegreatest extent in prostate tumor tissue relative to normal prostatetissue (approximately 20 fold difference). Significant differentalexpression was also observed in the testicular tumor tissue relative tonormal testicular tissue.

[0286] Characterization of the TRP-PLIK2 polypeptide of the presentinvention using antisense oligonucleotides directed against a portion ofthe TRP-PLIK2 encoding sequence led to the determination that it isinvolved in the modulation of the NFKB pathway, either directly orindirectly.

[0287] The upregulation of IkBa due to the downregulation of TRP-PLIK2places this transient receptor potential protein into a signallingpathway potentially involved in apoptotic events. This gives theopportunity to regulate downstream events via the activity of theprotein TRP-PLIK2 with antisense polynucleotides, polypeptides or lowmolecular chemicals with the potential of achieving a therapeutic effectin cancer, autoimmune diseases. In addition to cancer and immunologicaldisorders, NF-kB has significant roles in other diseases (Baldwin, A.S., J. Clin Invest. 107, :3-6 (2001)). NF-kB is a key factor in thepathophysiology of ischemia-reperfusion injury and heart failure (Valen,G., Yan. Z Q, Hansson G K, J. Am. Coll. Cardiol. 38, 307-14 (2001)).Furthermore, NF-kB has been found to be activated in experimental renaldisease (Guijarro C, Egido J., Kidney Int. 59, 415-425 (2001)). AsTRP-PLIK2 is highly expressed in kidney there is the potential of aninvolvement in renal diseases.

[0288] In preferred embodiments, TRP-PLIK2c polynucleotides andpolypeptides, including fragments thereof, are useful for treating,diagnosing, and/or ameliorating proliferative disorders, cancers,ischemia-reperfusion injury, heart failure, immuno compromisedconditions, HIV infection, and renal diseases.

[0289] Moreover, TRP-PLIK2c polynucleotides and polypeptides, includingfragments thereof, are useful for increasing NF-kB activity, increasingapoptotic events, and/or decreasing IkBa expression or activity levels.

[0290] In preferred embodiments, antagonists directed against TRP-PLIK2care useful for treating, diagnosing, and/or ameliorating autoimmunedisorders, disorders related to hyper immune activity, inflammatoryconditions, disorders related to aberrant acute phase responses,hypercongenital conditions, birth defects, necrotic lesions, wounds,organ transplant rejection, conditions related to organ transplantrejection, disorders related to aberrant signal transduction,proliferating disorders, cancers, HIV, and HIV propagation in cellsinfected with other viruses.

[0291] Moreover, antagonists directed against TRP-PLIK2c are useful fordecreasing NF-kB activity, decreasing apoptotic events, and/orincreasing IkBa expression or activity levels.

[0292] In preferred embodiments, agonists directed against TRP-PLIK2care useful for treating, diagnosing, and/or ameliorating autoimmunediorders, disorders related to hyper immune activity, hypercongenitalconditions, birth defects, necrotic lesions, wounds, disorders relatedto aberrant signal transduction, immuno compromised conditions, HIVinfection, proliferating disorders, and/or cancers.

[0293] Moreover, agonists directed against TRP-PLIK2c are useful forincreasing NF-kB activity, increasing apoptotic events, and/ordecreasing IkBa expression or activity levels.

[0294] The strong homology to transient receptor potential channels(TRP), combined with the predominate localized expression of theTRP-PLIK2 polypeptide in the lower gastrointestinal tract, specificallythe ileum, the rectum, the colon, the jejunum, and to a lesser extent inthe duodenum and stomach, suggests the TRP-PLIK2c polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing gastrointesinal diseases and/or disorders, which include, butare not limited to, ulcers, irritable bowel syndrome, inflammatory boweldisease, diarrhea, traveler's diarrhea, drug-related diarrhea polyps,absorption disorders, constipation, diverticulitis, vascular disease ofthe intestines, intestinal obstruction, intestinal infections,ulcerative colitis, Shigellosis, cholera, Crohn's Disease, amebiasis,enteric fever, Whipple's Disease, peritonitis, intrabdominal abcesses,hereditary hemochromatosis, gastroenteritis, viral gastroenteritis, foodpoisoning, mesenteric ischemia, mesenteric infarction, in addition to,metabolic diseases and/or disorders.

[0295] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosingsusceptibility to the following, non-limiting, gastrointestinalinfections: Salmonella infection, E. coli infection, E.coli 0157:H7infection, Shiga Toxin-producing E. coli infection, Campylobacterinfection (e.g., Campylobacter fetus, Campylobacter upsaliensis,Campylobacter hyointestinalis, Campylobacter lari, Campylobacter jejuni,Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum,Campylobacter rectus, Campylobacter curvus, Campylobacter sputorum,etc.), Heliobacter infection (e.g., Heliobacter cinaedi, Heliobacterfennelliae, etc.) Yersinia enterocolitica infection, Vibrio sp.Infection (e.g., Vibrio mimicus, Vibrio parahaemolyticus, Vibriofluvialis, Vibrio fumissii, Vibrio hollisae, Vibrio vulnificus, Vibrioalginolyticus, Vibrio metschnikovii, Vibrio damsela, Vibriocincinnatiensis, etc.) Aeromonas infection (e.g., Aeromonas hydrophila,Aeromonas sobira, Aeromonas caviae, etc.), Plesiomonas shigelliodesinfection, Giardia infection (e.g., Giardia lamblia, etc.),Cryptosporidium infection, Listeria infection, Entamoeba histolyticainfection, Rotavirus infection, Norwalk virus infection, Clostridiumdifficile infection, Clostriudium perfringens infection, Staphylococcusinfection, Bacillus infection, in addition to any other gastrointestinaldisease and/or disorder implicated by the causative agents listed aboveor elsewhere herein.

[0296] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of the TRP-PLIK2polypeptide in kidney tissue suggests the TRP-PLIK2c polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention ,slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0297] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trpl2 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H.,Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0298] Thus, the TRP-PLIK2c polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein.

[0299] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of the TRP-PLIK2polypeptide in bone marrow tissue suggests the TRP-PLIK2cpolynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing immune diseases and/or disorders.Representative uses are described in the “Immune Activity”,“Chemotaxis”, and “Infectious Disease” sections below, and elsewhereherein. Briefly, the strong expression in immune tissue indicates a rolein regulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.

[0300] The TRP-PLIK2c polypeptide may also be useful as a preventativeagent for immunological disorders including arthritis, asthma,immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis,granulomatous disease, inflammatory bowel disease, sepsis, acne,neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cellmediated cytotoxicity; immune reactions to transplanted organs andtissues, such as host-versus-graft and graft-versus-host diseases, orautoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma. The TRP-PLIK2 polypeptide may be useful for modulatingcytokine production, antigen presentation, or other processes, such asfor boosting immune responses, etc.

[0301] Moreover, the protein may represent a factor that influences thedifferentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, astissuemarkers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0302] Significantly, TRP-PLIK2c is believed to represent the first TRPfamily member expressed in bone marrow tissue.

[0303] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of TRP-PLIK intestis tissue emphasizes the potential utility for TRP-PLIK2cpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0304] In preferred embodiments, TRP-PLIK2c polynucleotides andpolypeptides including agonists, antagonists, and/or fragments thereof,have uses which include treating, diagnosing, prognosing, and/orpreventing the following, non-limiting, diseases or disorders of thetestis: spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The TRP-PLIK2cpolynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0305] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for TRP-PLIK2c polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0306] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0307] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I.,Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Birnbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0308] Thus, the TRP-PLIK2c polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein.

[0309] The predominate differential expression of TRP-PLIK2 in prostatetumor relative to normal prostate tissue strongly suggests TRP-PLIK2cpolynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, have uses which include treating, diagnosing,prognosing, and/or preventing prostate cancers and/or proliferativeconditions.

[0310] Alternatively, the tissue distribution of TRP-PLIK2 in liverindicates the protein product of the TRP-PLIK2c clone would be usefulfor the detection and treatment of liver disorders and cancers.Representative uses are described in the “Hyperproliferative Disorders”,“Infectious Disease”, and “Binding Activity” sections below, andelsewhere herein. Briefly, the protein can be used for the detection,treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis,liver metabolic diseases and conditions that are attributable to thedifferentiation of hepatocyte progenitor cells, cirrhosis, hepaticcysts, pyrogenic abscess, amebic abcess, hydatid cyst,cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma,hepatocellulae carcinoma, cholangiocarcinoma, angiosarcoma, andgranulomatous liver disease.

[0311] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosing thefollowing, non-limiting, hepatic infections: liver disease caused bysepsis infection, liver disease caused by bacteremia, liver diseasecaused by Pneomococcal pneumonia infection, liver disease caused byToxic shock syndrome, liver disease caused by Listeriosis, liver diseasecaused by Legionnaries' disease, liver disease caused by Brucellosisinfection, liver disease caused by Neisseria gonorrhoeae infection,liver disease caused by Yersinia infection, liver disease caused bySalmonellosis, liver disease caused by Nocardiosis, liver disease causedby Spirochete infection, liver disease caused by Treponema palliduminfection, liver disease caused by Brrelia burgdorferi infection, liverdisease caused by Leptospirosis, liver disease caused by Coxiellaburnetii infection, liver disease caused by Rickettsia richettsiiinfection, liver disease caused by Chlamydia trachomatis infection,liver disease caused by Chlamydia psittaci infection, in addition to anyother hepatic disease and/or disorder implicated by the causative agentslisted above or elsewhere herein.

[0312] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0313] In preferred embodiments, TRP-PLIK2c polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0314] In more preferred embodiments, TRP-PLIK2c polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0315] TRP-PLIK2c polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since TRP-PLIK2 is dominantly expressed in bone marrow, the TRP-PLIK2csplice variant may play an important role in regulating cytosolic Ca²⁺in immune system.

[0316] The TRP-PLIK2 gene maps to chromosome 9q21.2-22.1. This region islinked to amyotrophic lateral sclerosis with frontotemporal dementia,early-onset pulverulent cataract, infantile nephronophthisis,hypomagnesemia with secondary hypocalcemia and familial hemophagocyticlymphohistiocytosis. Therefore, agonists and/or antagonists of the novelTRP-PLIK2c splice variant can be used to treat diseases includingvarious forms of neuronal degeneration, neurogenic inflammation,allergy, immunodeficiency/excessive immune activation, visual defects,hearing disorder, pain, cancer, hypertension and other cardiovasculardiseases. In addition, the therapeutics may be useful in the treatmentof diseases associated with disturbances in Ca²⁺ homeostasis includingosteoporosis, hypercalciuric stone disease, and chronic renal failure.

[0317] In addition, TRP-PLIK2c polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ senstive proteins, the activation of Ca++ senstivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0318] The TRP-PLIK2c polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian bone marrow, kidney, testis, liver, small intestine, spinalcord, prostate, uterus, lung, lymph node, stomach, heart, brain, thymus,and pancreas, preferably human. TRP-PLIK2c polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, may be useful in diagnosing, treating, prognosing,and/or preventing immune, hematopoietic, renal, reproductive, hepatic,and/or proliferative diseases or disorders, particularly of the immunesystem.

[0319] In addition, antagonists of the TRP-PLIK2c polynucleotides andpolypeptides may have uses that include diagnosing, treating,prognosing, and/or preventing diseases or disorders related to transientreceptor potential channel activity, which may include immune,hematopoietic, renal, reproductive, hepatic, and/or proliferativediseases or disorders.

[0320] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those from CHAK1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the TRP-PLIK2cpolynucleotides, in addition to, other clones of the present invention,may be arrayed on microchips for expression profiling. Depending onwhich polynucleotide probe is used to hybridize to the slides, a changein expression of a specific gene may provide additional insight into thefunction of this gene based upon the conditions being studied. Forexample, an observed increase or decrease in expression levels when thepolynucleotide probe used comes from tissue that has been treated withknown immunoglobulin inhibitors, which include, but are not limited tothe drugs listed herein or otherwise known in the art, might indicate afunction in modulating immunoglobulin function, for example. In the caseof TRP-PLIK2c, bone marrow, kidney, testis, liver, small intestine,spinal cord, prostate, uterus, lung, lymph node, stomach, heart, brain,thymus, and/or pancrease, should be used to extract RNA to prepare theprobe.

[0321] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the TRP-PLIK2c gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:5 (FIGS. 3A-G).

[0322] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theTRP-PLIK2c, transforming yeast deficient in transient receptor potentialchannel activity with TRP-PLIK2c and assessing their ability to growwould provide convincing evidence the TRP-PLIK2c polypeptide hastransient receptor potential channel activity. Additional assayconditions and methods that may be used in assessing the function of thepolynucletides and polypeptides of the present invention are known inthe art, some of which are disclosed elsewhere herein.

[0323] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0324] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a bone marrow,kidney, testis, liver, small intestine, spinal cord, prostate, uterus,lung, lymph node, stomach, heart, brain, thymus, and/orpancrease-specific promoter), or it can be expressed at a specified timeof development using an inducible and/or a developmentally regulatedpromoter.

[0325] In the case of TRP-PLIK2c transgenic mice or rats, if nophenotype is apparent in normal growth conditions, observing theorganism under diseased conditions (immune, hematopoietic, renal,reproductive, hepatic, or proliferative disorders, etc.) may lead tounderstanding the function of the gene. Therefore, the application ofantisense and/or sense methodology to the creation of transgenic mice orrats to refine the biological function of the polypeptide is encompassedby the present invention.

[0326] In preferred embodiments, the following N-terminal TRP-PLIK2cdeletion polypeptides are encompassed by the present invention:M1-L1939, 12-L1939, 13-L1939, L4-L1939, S5-L1939, K6-L1939, S7-L1939,Q8-L1939, K9-L1939, S10-L1939, W11-L1939, S5-L1939, K13-L1939,G14-L1939, V15-L1939, F16-L1939, D17-L1939, K18-L1939, R19-L1939,E20-L1939, C21-L1939, S22-L1939, T23-L1939, 124-L1939, 125-L1939,P26-L1939, S27-L1939, S28-L1939, K29-L1939, N30-L1939, P31-L1939,H32-L1939, R33-L1939, C34-L1939, T35-L1939, P36-L1939, V37-L1939,C38-L1939, Q39-L1939, V40-L1939, C41-L1939, Q42-L1939, N43-L1939,L44-L1939, 145-L1939, R46-L1939, C47-L1939, Y48-L1939, C49-L1939,G50-L1939, R51-L1939, L52-L1939, 153-L1939, G54-L1939, D55-L1939,H56-L1939, A57-L1939, G58-L1939, 159-L1939, D60-L1939, Y61-L1939,S62-L1939, W63-L1939, T64-L1939, 165-L1939, S66-L1939, A67-L1939,A68-L1939, K69-L1939, G70-L1939, K71-L1939, E72-L1939, S73-L1939,E74-L1939, Q75-L1939, W76-L1939, S77-L1939, V78-L1939, E79-L1939,K80-L1939, H81-L1939, T82-L1939, T83-L1939, K84-L1939, S85-L1939,P86-L1939, T87-L1939, D88-L1939, T89-L1939, F90-L1939, G91-L1939,T92-L1939, 193-L1939, N94-L1939, F95-L1939, Q96-L1939, D97-L1939,G98-L1939, E99-L1939, H100-L1939, T101-L1939, H102-L1939, H103-L1939,A104-L1939, K105-L1939, Y106-L1939, 1107-L1939, R108-L1939, T109-L1939,S100-L1939, Ylil-L1939, D112-L1939, T113-L1939, K114-L1939, L115-L1939,D116-L1939, H117-L1939, L118-L1939, L119-L1939, H120-L1939, L121-L1939,M122-L1939, L123-L1939, K124-L1939, E125-L1939, W126-L1939, K127-L1939,M128-L1939, E129-L1939, L130-L1939, P131-L1939, K132-L1939, L133-L1939,V134-L1939, 1135-L1939, S136-L1939, V137-L1939, H138-L1939, G139-L1939,G140-L1939, 1141-L1939, Q142-L1939, N143-L1939, F144-L1939, T145-L1939,M146-L1939, P147-L1939, S148-L1939, K149-L1939, F150-L1939, K151-L1939,E152-L1939, 1153-L1939, F154-L1939, S155-L1939, Q156-L1939, G157-L1939,L158-L1939, V159-L1939, K160-L1939, A161-L1939, A162-L1939, E163-L1939,T164-L1939, T165-L1939, G166-L1939, A167-L1939, W168-L1939, 1169-L1939,1170-L1939, T171-L1939, E172-L1939, G173-L1939, 1174-L1939, N175-L1939,T176-L1939, G177-L1939, V178-L1939, S179-L1939, K180-L1939, H181-L1939,V182-L1939, G183-L1939, D184-L1939, A185-L1939, L186-L1939, K187-L1939,S188-L1939, H189-L1939, S190-L1939, S191-L1939, H192-L1939, S193-L1939,L194-L1939, R195-L1939, K196-L1939, 1197-L1939, W198-L1939, T199-L1939,V200-L1939, G201-L1939, 1202-L1939, P203-L1939, P204-L1939, W205-L1939,G206-L1939, V207-L1939, 1208-L1939, E209-L1939, N210-L1939, Q211-L1939,R212-L1939, D213-L1939, L214-L1939, 1215-L1939, G216-L1939, K217-L1939,D218-L1939, V219-L1939, V220-L1939, C221-L1939, L222-L1939, Y223-L1939,Q224-L1939, T225-L1939, L226-L1939, D227-L1939, N228-L1939, P229-L1939,L230-L1939, S231-L1939, K232-L1939, L233-L1939, T234-L1939, T235-L1939,L236-L1939, N237-L1939, S238-L1939, M239-L1939, H240-L1939, S241-L1939,H242-L1939, F243-L1939, 1244-L1939, L245-L1939, S246-L1939, D247-L1939,D248-L1939, G249-L1939, T250-L1939, V251-L1939, G252-L1939, K253-L1939,Y254-L1939, G255-L1939, N256-L1939, E257-L1939, M258-L1939, K259-L1939,L260-L1939, R261-L1939, R262-L1939, N263-L1939, L264-L1939, E265-L1939,K266-L1939, Y267-L1939, L268-L1939, S269-L1939, L270-L1939, Q271-L1939,K272-L1939, 1273-L1939, H274-L1939, C275-L1939, R276-L1939, S277-L1939,R278-L1939, Q279-L1939, G280-L1939, V281-L1939, P282-L1939, V283-L1939,V284-L1939, G285-L1939, L286-L1939, V287-L1939, V288-L1939, E289-L1939,G290-L1939, G291-L1939, P292-L1939, N293-L1939, V294-L1939, 1295-L1939,L296-L1939, S297-L1939, V298-L1939, W299-L1939, E300-L1939, T301-L1939,V302-L1939, K303-L1939, D304-L1939, K305-L1939, D306-L1939, P307-L1939,V308-L1939, V309-L1939, V310-L1939, C311-L1939, E312-L1939, G313-L1939,T314-L1939, G315-L1939, R316-L1939, A317-L1939, A318-L1939, D319-L1939,L320-L1939, L321-L1939, A322-L1939, F323-L1939, T324-L1939, H325-L1939,K326-L1939, H327-L1939, L328-L1939, A329-L1939, D330-L1939, E331-L1939,G332-L1939, M333-L1939, L334-L1939, R335-L1939, P336-L1939, Q337-L1939,V338-L1939, K339-L1939, E340-L1939, E341-L1939, 1342-L1939, 1343-L1939,C344-L1939, M345-L1939, 1346-L1939, Q347-L1939, N348-L1939, T349-L1939,F350-L1939, N351-L1939, F352-L1939, S353-L1939, L354-L1939, K355-L1939,Q356-L1939, S357-L1939, K358-L1939, H359-L1939, L360-L1939, F361-L1939,Q362-L1939, 1363-L1939, L364-L1939, M365-L1939, E366-L1939, C367-L1939,M368-L1939, V369-L1939, H370-L1939, R371-L1939, D372-L1939, C373-L1939,1374-L1939, T375-L1939, 1376-L1939, F377-L1939, D378-L1939, A379-L1939,D380-L1939, S381-L1939, E382-L1939, E383-L1939, Q384-L1939, Q385-L1939,D386-L1939, L387-L1939, D388-L1939, L389-L1939, A390-L1939, 1391-L1939,L392-L1939, T393-L1939, A394-L1939, L395-L1939, L396-L1939, K397-L1939,G398-Ll939, T399-L1939, N400-L1939, L401-L1939, S402-L1939, A403-L1939,S404-L1939, E405-L1939, Q406-L1939, L407-L1939, N408-L1939, L409-L1939,A410-L1939, M411-L1939, A412-L1939, W413-L1939, D414-L1939, R415-L1939,V416-L1939, D417-L1939, 1418-L1939, A419-L1939, K420-L1939, K421-L1939,H422-L1939, 1423-L1939, L424-L1939, 1425-L1939, Y426-L1939, E427-L1939,Q428-L1939, H429-L1939, W430-L1939, K431-L1939, P432-L1939, D433-L1939,A434-L1939, L435-L1939, E436-L1939, Q437-L1939, A438-L1939, M439-L1939,S440-L1939, D441-L1939, A442-L1939, L443-L1939, V444-L1939, M445-L1939,D446-L1939, R447-L1939, V448-L1939, D449-L1939, F450-L1939, V451-L1939,K452-L1939, L453-L1939, IA54-L1939, 1455-L1939, E456-L1939, Y457-L1939,G458-L1939, V459-L1939, N460-L1939, L461-L1939, H462-L1939, R463-L1939,F464-L1939, L465-L1939, T466-L1939, 1467-L1939, P468-L1939, R469-L1939,L470-L1939, E471-L1939, E472-L1939, L473-L1939, Y474-L1939, N475-L1939,T476-L1939, K477-L1939, Q478-L1939, G479-L1939, P480-L1939, T481-L1939,N482-L1939, T483-L1939, L484-L1939, L485-L1939, H486-L1939, H487-L1939,L488-L1939, V489-L1939, Q490-L1939, D491-L1939, V492-L1939, K493-L1939,and/or Q494-L1939 of SEQ ID NO:6. Polynucleotide sequences encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these N-terminal TRP-PLIK2c deletion polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

[0327] In preferred embodiments, the following C-terminal TRP-PLIK2cdeletion polypeptides are encompassed by the present invention:M1-L1939, M1-Q1938, M1-M1937, M1-D1936, M1-D1935, M1-E1934, M1-P1933,M1-S1932, M1-N1931, M1-R1930, M1-G1929, M1-T1928, M1-E1927, M1-R1926,M1-A1925, M1-P1924, M1-P1923, M1-E1922, M1-E1921, M1-A1920, M1-S1919,M1-E1918, M1-I1917, M1-K1916, M1-I1915, M1-E1914, M1-L1913, M1-G1912,M1-F1911, M1-T1910, M1-S1909, M1-N1908, M1-I1907, M1-R1906, M1-E1905,M1-P1904, M1-S1903, M1-Y1902, M1-D1901, M1-N1900, M1-R1899, M1-K1898,M1-L1897, M1-D1896, M1-P1895, M1-L1894, M1-K1893, M1-L1892, M1-Kl891,M1-R1890, M1-C1889, M1-C1888, M1-S1887, M1-N1886, M1-C1885, M1-H1884,M1-H1883, M1-K1882, M1-A1881, M1-I1880, M1-F1879, M1-N1878, M1-R1877,M1-11876, M1-A1875, M1-D1874, M1-E1873, M1-G1872, M1-L1871, M1-N1870,M1-A1869, M1-P1868, M1-G1867, M1-F1866, M1-V1865, M1-M1864, M1-G1863,M1-R1862, M1-S1861, M1-Q1860, M1-K1859, M1-V1858, M1-E1857, M1-P1856,M1-K1855, M1-I1854, M1-V1853, M1-S1852, M1-P1851, M1-D1850, M1-T1849,M1-L1848, M1-N1847, M1-E1846, M1-G1845, M1-V1844, M1-G1843, M1-Q1842,M1-L1841, M1-D1840, M1-L1839, M1-V1838, M1-L1837, M1-L1836, M1-E1835,M1-G1834, M1-R1833, M1-T1832, M1-Y1831, M1-E1830, M1-Y1829, M1-T1828,M1-W1827, M1-H1826, M1-S1825, M1-F1824, M1-A1823, M1-L1822, M1-M1821,M1-L1820, M1-E1819, M1-E1818, M1-L1817, M1-T1816, M1-N1815, M1-T1814,M1-P1813, M1-T1812, M1-I1811, M1-E1810, M1-D1809, M1-G1808, M1-N1807,M1-N1806, M1-N1805, M1-N1804, M1-Y1803, M1-K1802, M1-R1801, M1-F1800,M1-E1799, M1-G1798, M1-T1797, M1-M1796, M1-Y1795, M1-K1794, M1-E1793,M1-I1792, M1-T1791, M1-L1790, M1-W1789, M1-Q1788, M1-N1787, M1-A1786,M1-S1785, M1-H1784, M1-C1783, M1-Y1782, M1-I1781, M1-L1780, M1-F1779,M1-V1778, M1-E1777, M1-L1776, M1-F1775, M1-R1774, M1-P1773, M1-T1772,M1-Y1771, M1-P1770, M1-I1769, M1-T1768, M1-Q1767, M1-P1766, M1-K1765,M1-V1764, M1-Q1763, M1-N1762, M1-F1761, M1-T1760, M1-Y1759, M1-I1758,M1-L1757, M1-K1756, M1-Q1755, M1-A1754, M1-A1753, M1-R1752, M1-Q1751,M1-Q1750, M1-Q1749, M1-I1748, M1-E1747, M1-R1746, M1-L1745, M1-C1744,M1-L1743, M1-H1742, M1-L1741, M1-V1740, M1-T1739, M1-S1738, M1-E1737,M1-Q1736, M1-F1735, M1-I1734, M1-K1733, M1-H1732, M1-W1731, M1-T1730,M1-R1729, M1-V1728, M1-V1727, M1-E1726, M1-P1725, M1-L1724, M1-F1723,M1-S1722, M1-K1721, M1-V1720, M1-I1719, M1-F1718, M1-V1717, M1-Q1716,M1-G1715, M1-P1714, M1-K1713, M1-L1712, M1-I1711, M1-D1710, M1-D1709,M1-E1708, M1-S1707, M1-W1706, M1-T1705, M1-S1704, M1-V1703, M1-V1702,M1-R1701, M1-M1700, M1-A1699, M1-K1698, M1-R1697, M1-L1696, M1-G1695,M1-G1694, M1-D1693, M1-M1692, M1-E1691, M1-E1690, M1-R1689, M1-S1688,M1-L1687, M1-V1686, M1-Q1685, M1-I1684, M1-M1683, M1-A1682, M1-A1681,M1-R1680, M1-G1679, M1-R1678, M1-Q1677, M1-S1676, M1-W1675, M1-S1674,M1-S1673, M1-M1672, M1-S1671, M1-K1670, M1-D1669, M1-L1668, M1-N1667,M1-L1666, M1-P1665, M1-S1664, M1-S1663, M1-E1662, M1-E1661, M1-L1660,M1-R1659, M1-Y1658, M1-V1657, M1-T1656, M1-I1655, M1-E1654, M1-E1653,M1-G1652, M1-A1651, M1-F1650, M1-L1649, M1-Q1648, M1-V1647, M1-P1646,M1-T1645, M1-F1644, M1-P1643, M1-I1642, M1-T1641, M1-Q1640, M1-S1639,M1-L1638, M1-R1637, M1-M1636, M1-L1635, M1-N1634, M1-N1633, M1-R1632,M1-E1631, M1-I1630, M1-A1629, M1-S1628, M1-Y1627, M1-H1626, M1-H1625,M1-H1624, M1-P1623, M1-E1622, M1-Q1621, M1-P1620, M1-S1619, M1-K1618,M1-L1617, M1-S1616, M1-A1615, M1-S1614, M1-I1613, M1-K1612, M1-D1611,M1-V1610, M1-G1609, M1-I1608, M1-S1607, M1-S1606, M1-K1605, M1-L1604,M1-L1603, M1-S1602, M1-N1601, M1-R1600, M1-N1599, M1-L1598, M1-N1597,M1-T1596, M1-S1595, M1-R1594, M1-S1593, M1-N1592, M1-W1591, M1-L1590,M1-S1589, M1-N1588, M1-K1587, M1-S1586, M1-L1585, M1-D1584, M1-E1583,M1-Q1582, M1-S1581, M1-Q1580, M1-K1579, M1-L1578, M1-Y1577, M1-D1576,M1-S1575, M1-I1574, M1-Q1573, M1-I1572, M1-A1571, M1-C1570, M1-Q1569,M1-G1568, M1-I1567, M1-E1566, M1-K1565, M1-T1564, M1-K1563, M1-M1562,M1-K1561, M1-Q1560, M1-H1559, M1-I1558, M1-Y1557, M1-P1556, M1-E1555,M1-V1554, M1-G1553, M1-T1552, M1-H1551, M1-S1550, M1-F1549, M1-K1548,M1-S1547, M1-V1546, M1-T1545, M1-F1544, M1-W1543, M1-N1542, M1-K1541,M1-S1540, M1-Y1539, M1-E1538, M1-E1537, M1-E1536, M1-S1535, M1-I1534,M1-S1533, M1-N1532, M1-E1531, M1-G1530, M1-P1529, M1-E1528, M1-P1527,M1-N1526, M1-L1525, M1-Q1524, M1-D1523, M1-S1522, M1-Q1521, M1-S1520,M1-C1519, M1-A1518, M1-N1517, M1-V1516, M1-T1515, M1-11514, M1-I1513,M1-P1512, M1-V1511, M1-Q1510, M1-L1509, M1-G1508, M1-Q1507, M1-T1506,M1-N1505, M1-K1504, M1-K1503, M1-K1502, M1-K1501, M1-S1500, M1-L1499,M1-R1498, M1-R1497, M1-D1496, M1-K1495, M1-T1494, M1-L1493, M1-M1492,M1-K1491, M1-A1490, M1-K1489, M1-V1488, M1-W1487, M1-A1486, M1-G1485,M1-Q1484, M1-G1483, M1-I1482, M1-E1481, M1-S1480, M1-S1479, M1-G1478,M1-S1477, M1-L1476, M1-N1475, M1-K1474, M1-I1473, M1-K1472, M1-C1471,M1-I1470, M1-K1469, M1-M1468, M1-L1467, M1-K1466, M1-E1465, M1-E1464,M1-K1463, M1-H1462, M1-F1461, M1-R1460, M1-F1459, M1-S1458, M1-H1457,M1-S1456, M1-R1455, M1-A1454, M1-F1453, M1-P1452, M1-R1451, M1-Y1450,M1-R1449, M1-R1448, M1-L1447, M1-P1446, M1-N1445, M1-I1444, M1-W1443,M1-F1442, M1-S1441, M1-T1440, M1-N1439, M1-P1438, M1-Q1437, M1-L1436,M1-W1435, M1-P1434, M1-G1433, M1-V1432, M1-E1431, M1-S1430, M1-C1429,M1-E1428, M1-S1427, M1-S1426, M1-Q1425, M1-A1424, M1-S1423, M1-R1422,M1-T1421, M1-S1420, M1-N1419, M1-D1418, M1-S1417, M1-L1416, M1-S1415,M1-S1414, M1-D1413, M1-Q1412, M1-A1411, M1-Q1410, M1-K1409, M1-Q1408,M1-H1407, M1-Q1406, M1-E1405, M1-S1404, M1-R1403, M1-S1402, M1-S1401,M1-D1400, M1-S1399, M1-D1398, M1-C1397, M1-T1396, M1-S1395, M1-P1394,M1-L1393, M1-C1392, M1-T1391, M1-Q1390, M1-W1389, M1-K1388, M1-K1387,M1-K1386, M1-I1385, M1-S1384, M1-F1383, M1-V1382, M1-G1381, M1-T1380,M1-E1379, M1-D1378, M1-G1377, M1-E1376, M1-S1375, M1-F1374, M1-A1373,M1-W1372, M1-N1371, M1-V1370, M1-Y1369, M1-G1368, M1-G1367, M1-G1366,M1-T1365, M1-Q1364, M1-M1363, M1-I1362, M1-K1361, M1-A1360, M1-Q1359,M1-S1358, M1-L1357, M1-P1356, M1-S1355, M1-S1354, M1-M1353, M1-T1352,M1-M1351, M1-P1350, M1-E1349, M1-P1348, M1-T1347, M1-C1346, M1-S1345,M1-L1344, M1-T1343, M1-P1342, M1-L1341, M1-V1340, M1-Q1339, M1-E1338,M1-A1337, M1-K1336, M1-D1335, M1-Q1334, M1-G1333, M1-D1332, M1-L1331,M1-L1330, M1-H1329, M1-A1328, M1-I1327, M1-P1326, M1-E1325, M1-H1324,M1-K1323, M1-E1322, M1-K1321, M1-P1320, M1-E1319, M1-D1318, M1-V1317,M1-S1316, M1-A1315, M1-W1314, M1-D1313, M1-S1312, M1-V1311, M1-V1310,M1-P1309, M1-T1308, M1-Q1307, M1-G1306, M1-T1305, M1-L1304, M1-H1303,M1-V1302, M1-L1301, M1-V1300, M1-E1299, M1-T1298, M1-Q1297, M1-I1296,M1-D1295, M1-Q1294, M1-E1293, M1-T1292, M1-A1291, M1-L1290, M1-V1289,M1-D1288, M1-P1287, M1-V1286, M1-S1285, M1-P1284, M1-R1283, M1-S1282,M1-L1281, M1-P1280, M1-L1279, M1-V1278, M1-T1277, M1-E1276, M1-A1275,M1-S1274, M1-F1273, M1-P1272, M1-V1271, M1-R1270, M1-K1269, M1-L1268,M1-N1267, M1-S1266, M1-P1265, M1-V1264, M1-L1263, M1-L1262, M1-F1261,M1-Q1260, M1-G1259, M1-Y1258, M1-K1257, M1-S1256, M1-H1255, M1-A1254,M1-Q1253, M1-R1252, M1-N1251, M1-P1250, M1-S1249, M1-V1248, M1-G1247,M1-S1246, M1-V1245, M1-V1244, M1-I1243, M1-S1242, M1-S1241, M1-Q1240,M1-T1239, M1-E1238, M1-Q1237, M1-R1236, M1-E1235, M1-Q1234, M1-D1233,M1-N1232, M1-R1231, M1-V1230, M1-N1229, M1-T1228, M1-A1227, M1-E1226,M1-R1225, M1-K1224, M1-S1223, M1-N1222, M1-T1221, M1-I1220, M1-E1219,M1-L1218, M1-L1217, M1-A1216, M1-G1215, M1-R1214, M1-Q1213, M1-V1212,M1-R1211, M1-P1210, M1-P1209, M1-H1208, M1-R1207, M1-G1206, M1-G1205,M1-A1204, M1-L1203, M1-S1202, M1-R1201, M1-L1200, M1-L1199, M1-S1198,M1-S1197, M1-P1196, M1-M1195, M1-S1194, M1-Y1193, M1-Y1192, M1-Q1191,M1-Y1190, M1-K1189, M1-K1188, M1-E1187, M1-G1186, M1-A1185, M1-I1184,M1-E1183, M1-M1182, M1-S1181, M1-G1180, M1-L1179, M1-V1178, M1-E1177,M1-A1176, M1-C1175, M1-I1174, M1-V1173, M1-N1172, M1-S1171, M1-W1170,M1-S1169, M1-H1168, M1-P1167, M1-L1166, M1-K1165, M1-K1164, M1-C1163,M1-T1162, M1-S1161, M1-H1160, M1-K1159, M1-R1158, M1-K1157, M1-A1156,M1-L1155, M1-L1154, M1-A1153, M1-E1152, M1-D1151, M1-E1150, M1-Q1149,M1-L1148, M1-T1147, M1-D1146, M1-V1145, M1-A1144, M1-S1143, M1-L1142,M1-V1141, M1-K1140, M1-L1139, M1-T1138, M1-D1137, M1-V1136, M1-T1135,M1-L1134, M1-A1133, M1-S1132, M1-L1131, M1-D1130, M1-Q1129, M1-L1128,M1-H1127, M1-G1126, M1-V1125, M1-Q1124, M1-S1123, M1-D1122, M1-L1121,M1-S1120, M1-L119, M1-L1118, M1-S1117, M1-D1116, M1-K1115, M1-I1114,M1-F1113, M1-S1112, M1-V1111, M1-K1110, M1-E1109, M1-N1108, M1-M1107,M1-E1106, M1-K1105, M1-L1104, M1-Q1103, M1-F1102, M1-Y1101, M1-M1100,M1-E1099, M1-T1098, M1-V1097, M1-R1096, M1-E1095, M1-S1094, M1-T1093,M1-V1092, M1-R1091, M1-I1090, M1-R1089, M1-E1088, M1-E1087, M1-C1086,M1-S1085, M1-C1084, M1-N1083, M1-V1082, M1-D1081, M1-E1080, M1-M1079,M1-K1078, M1-E1077, M1-H1076, M1-F1075, M1-Y1074, M1-K1073, M1-E1072,M1-V1071, M1-C1070, M1-Q1069, M1-E1068, M1-E1067, M1-F1066, M1-D1065,M1-H1064, M1-L1063, M1-K1062, M1-K1061, M1-L1060, M1-D1059, M1-E1058,M1-K1057, M1-S1056, M1-L1055, M1-Y1054, M1-L1053, M1-K1052, M1-L1051,M1-G1050, M1-V1049, M1-D1048, M1-G1047, M1-E1046, M1-E1045, M1-Q1044,M1-D1043, M1-H1042, M1-P1041, M1-A1040, M1-R1039, M1-H1038, M1-C1037,M1-C1036, M1-L1035, M1-R1034, M1-R1033, M1-L1032, M1-L1031, M1-L1030,M1-G1029, M1-V1028, M1-H1027, M1-S1026, M1-L1025, M1-L1024, M1-I1023,M1-L1022, M1-P1021, M1-P1020, M1-P1019, M1-L1018, M1-W1017, M1-P1016,M1-K1015, M1-E1014, M1-H1013, M1-Y1012, M1-T1011, M1-M1000, M1-I1009,M1-Y1008, M1-R1007, M1-Y1006, M1-R1005, M1-N1004, M1-Y1003, M1-K1002,M1-W1001, M1-L1000, M1-N999, M1-N998, M1-S997, M1-1996, M1-S995,M1-E994, M1-M993, M1-D992, M1-L991, M1-Y990, M1-V989, M1-N988, M1-N987,M1-F986, M1-F985, M1-A984, M1-1983, M1-L982, M1-L981, M1-N980, M1-V979,M1-M978, M1-1977, M1-1976, M1-Y975, M1-Q974, M1-V973, M1-F972, M1-L971,M1-Y970, M1-V969, M1-A968, M1-Q967, M1-L966, M1-F965, M1-P964, M1-T963,M1-L962, M1-F961, M1-S960, M1-G959, M1-P958, M1-P957, M1-C956, M1-S955,M1-P954, M1-Q953, M1-S952, M1-S951, M1-C950, M1-V949, M1-D948, M1-1947,M1-E946, M1-G945, M1-A944, M1-Y943, M1-V942, M1-E941, M1-G940, M1-Y939,M1-1938, M1-M937, M1-W936, M1-Y935, M1-P934, M1-E933, M1-F932, M1-V931,M1-1930, M1-D929, M1-R928, M1-A927, M1-L926, M1-S925, M1-W924, M1-S923,M1-P922, M1-P921, M1-E920, M1-K919, M1-P918, M1-S917, M1-L916, M1-I915,M1-A914, M1-K913, M1-R912, M1-A911, M1-V910, M1-G909, M1-F908, M1-S907,M1-L906, M1-L905, M1-V904, M1-1903, M1-A902, M1-M901, M1-1900, M1-1899,M1-V898, M1-1897, M1-Y896, M1-F895, M1-M894, M1-N893, M1-A892, M1-T891,M1-M890, M1-K889, M1-A888, M1-1887, M1-M886, M1-T885, M1-V884, M1-Y883,M1-P882, M1-G881, M1-A880, M1-H879, M1-Q878, M1-N877, M1-V876, M1-A875,M1-F874, M1-F873, M1-D872, M1-L871, M1-L870, M1-R869, M1-S868, M1-F867,M1-W866, M1-F865, M1-1864, M1-1863, M1-D862, M1-I861, M1-C860, M1-Y859,M1-I858, M1-L857, M1-R856, M1-G855, M1-A854, M1-T853, M1-H852, M1-F851,M1-P850, M1-P849, M1-D848, M1-G847, M1-W846, M1-R845, M1-L844, M1-V843,M1-F842, M1-G841, M1-A840, M1-S839, M1-F838, M1-L837, M1-G836, M1-835,M1-A834, M1-V833, M1-T832, M1-E831, M1-T830, M1-L829, M1-N828, M1-W827,M1-Y826, M1-E825, M1-S824, M1-I823, M1-W822, M1-V821, M1-K820, M1-V819,M1-K818, M1-Q817, M1-T816, M1-F815, M1-K814, M1-G813, M1-P812, M1-E811,M1-S810, M1-I809, M1-C808, M1-1807, M1-E806, M1-R805, M1-V804, M1-V803,M1-E802, M1-1801, M1-A800, M1-N799, M1-T798, M1-F797, M11-796, M1-Y795,M1-I794, M1-S793, M1-V792, M1-L791, M1 -W790, M1-E789, M1-Q788, M1-V787,M1-S786, M1-P785, M1-Q784, M1-P783, M1-Q782, M1-M781, M1-E780, M1-V779,M1-L778, M1-V777, M1-T776, M1-Y775, M1-T774, M1-F773, M1-L772, M1-M771,M1-L770, M1-F769, M1-A768, M1-L767, M1-Y766, M1-A765, M1-M764, M1-T763,M1-Y762, M1-F761, M1-W760, M1-F759, M1-K758, M1-V757, M1-I756, M1-P755,M1-A754, M1-S753, M1-Y752, M1-F751, M1-E750, M1-Y749, M1-V748, M1-K747,M1-R746, M1-T745, M1-W744, M1-P743, M1-L742, M1-H74 1, M1-Q740, M1-H739,M1-G738, M1-S737, M1-E736, M1-L735, M1-G734, M1-F733, M1-H732, M1-Q73 1,M1-N730, M1-E729, M1-D728, M1-L727, M1-K726, M1-E725, M1-D724, M1-H723,M1-G722, M1-R721, M1-E720, M1-L719, M1-D718, M1-Y717, M1-E716, M1-K715,M1-V714, M1-S713, M1-A712, M1-S711, M1-E710, M1-K709, M1-S708, M1-S707,M1-S706, M1-A705, M1-N704, M1-Q703, M1-D702, M1-S701, M1-Y700, M1-Y699,M1-W698, M1-M697, M1-F696, M1-Q695, M1-F-694, M1-D693, M1-Q692, M1-S691,M1-Q690, M1-P689, M1-V688, M1-H687, M1-S686, M1-M685, M1-E684, M1-A683,M1-K682, M1-S68 1, M1-K680, M1-F679, M1-E678, M1-L677, M1-T676, M1-L675,M1-674, M1-T673, M1-P672, M1-P671, M1-L670, M1-I669, M1-I668, M1-S667,M1-I666, M1-I665, M1-I664, M1-K663, M1-L662, M1-W661, M1-S660, M1-N659,M1-K658, M1-R657, M1-M656, M1-K655, M1-L654, M1-R653, M1-G652, M1-M651,M1-W650, M1-M649, M1-D648, M1-T647, M1-L646, M1-L645, M1-M644, M1-Q643,M1-T642, M1-C641, M1-T640, M1-H639, M1-S638, M1-V637, M1-F636, M1-P635,M1-R634, M1-L633, M1-G632, M1-G631, M1-S630, M1-V629, M1-A628, M1-L627,M1-K626, M1-L625, M1-C624, M1-T623, M1-S622, M1-N621, M1-S620, M1-W619,M1-N618, M1-R617, M1-L616, M1-E615, M1-Y614, M1-T613, M1-L612, M1-L611,M1-T610, M1-M609, M1-A608, M1-M607, M1-R606, M1-E605, M1-N604, M1-Q603,M1-K602, M1-F601, M1-A600, M1-K599, M1-E598, M1-L597, M1-L596, M1-D595,M1-L594, M1-A593, M1-L592, M1-Q591, M1-G590, M1-F589, M1-Q588, M1-K587,M1-S586, M1-Y585, M1-N584, M1-K583, M1-L582, M1-E581, M1-E580, M1-S579,M1-A578, M1-D577, M1-D576, M1-V575, M1-M574, M1-H573, M1-S572, M1-E571,M1-K570, M1-A569, M1-E568, M1-H567, M1-A566, M1-M565, M1-A564, M1-R563,M1-Y562, M1-L561, M1-1560, M1-C559, M1-A558, M1-1557, M1-V556, M1-A555,M1-K554, M1-V553, M1-T552, M1-A551, M1-E550, M1-E549, M1-G548, M1-H547,M1-Q546, M1-W545, M1-F544, M1-F543, M1-M542, M1-A541, M1-M540, M1-K539,M1-Q538, M1-R537, M1-K536, M1-M535, M1-L534, M1-V533, M1-A532, M1-W531,M1-V530, M1-L529, M1-L528, M1-D527, M1-N526, M1-Y525, M1-P524, M1-Y523,M1-L522, M1-F521, M1-G520, M1-T519, M1-S518, M1-E517, M1-P516, M1-D515,M1-D514, M1-S513, M1-V512, M1-N511, M1-Q510, M1-E509, M1-K508, M1-S507,M1-K506, M1-K505, M1-R504, M1-S503, M1-K502, M1-H501, M1-L500, M1-V499,M1-1498, M1-S497, M1-K496, and/or M1-E495 of SEQ ID NO:6. Polynucleotidesequences encoding these polypeptides are also provided. The presentinvention also encompasses the use of these C-terminal TRP-PLIK2cdeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0328] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the TRP-PLIK2c polypeptide (e.g., any combination ofboth N- and C- terminal TRP-PLIK2c polypeptide deletions) of SEQ IDNO:6. For example, internal regions could be defined by the equation:amino acid NX to amino acid CX, wherein NX refers to any N-terminaldeletion polypeptide amino acid of TRP-PLIK2c (SEQ ID NO:6), and whereCX refers to any C-terminal deletion polypeptide amino acid ofTRP-PLIK2c (SEQ ID NO:6). Polynucleotides encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese polypeptides as an immunogenic and/or antigenic epitope asdescribed elsewhere herein.

[0329] The TRP-PLIK2c polypeptides of the present invention weredetermined to comprise several phosphorylation sites based upon theMotif algorithm (Genetics Computer Group, Inc.). The phosphorylation ofsuch sites may regulate some biological activity of the TRP-PLIK2cpolypeptide. For example, phosphorylation at specific sites may beinvolved in regulating the proteins ability to associate or bind toother molecules (e.g., proteins, ligands, substrates, DNA, etc.). In thepresent case, phosphorylation may modulate the ability of the TRP-PLIK2cpolypeptide to associate with other potassium channel alpha subunits,beta subunits, or its ability to modulate potassium channel function.

[0330] The TRP-PLIK2c polypeptide was predicted to comprise twenty eightPKC phosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. . . .260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0331] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: IILSKSQKSWIKG(SEQ ID NO:165), STIIPSSKNPHRC (SEQ ID NO:166), SVEKHTTKSPTDT (SEQ IDNO:167), SHSSHSLRKIWTV (SEQ ID NO:168), LSVWETVKDKDPV (SEQ ID NO:169),VVCEGTGRAADLL (SEQ ID NO:170), DLLAFTHKHLADE (SEQ ID NO:171),NTFNFSLKQSKHL (SEQ ID NO:172), IVLHKSRKKSKEQ (SEQ ID NO:173),HGEEATVKAVIAC (SEQ ID NO:174), DQNASSSKESASV (SEQ ID NO:175),SKESASVKEYDLE (SEQ ID NO:176), QHLPWTRKVYEFY (SEQ ID NO:177),EPGKFTQKVKVWI (SEQ ID NO:178), RKAILSPKEPPSW (SEQ ID NO:179),RIRVTSERVTEMY (SEQ ID NO:180), ALTVDTLKVLSAV (SEQ ID NO:181),KRKHSTCKKLPHS (SEQ ID NO:182), LEITNSKREATNV (SEQ ID NO:183),ETGVFSIKKKWQT (SEQ ID NO:184), TCDSDSSRSEQHQ (SEQ ID NO:185),SLSDNSTRSAQSS (SEQ ID NO:186), FARSHSFRFHKEE (SEQ I) NO:187),KDRRLSKKKKNTQ (SEQ ID NO:188), DKISASLKSPQEP (SEQ ID NO:189),SMSSWSQRGRAAM (SEQ ID NO:190), QTIPYTPRFLEVF (SEQ ID NO:191), and/orPPARETGRNSPED (SEQ ID NO:192). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these TRP-PLIK2c PKC phosphorylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0332] The present invention also encompasses immunogenic and/orantigenic epitopes of the TRP-PLIK2c polypeptide.

[0333] The TRP-PLIK2c polypeptide has been shown to comprise fifteenglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0334] Asparagine phosphorylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. . .. 265:11397-I1404(1990).

[0335] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:HGGIQNFTMPSKFK (SEQ ID NO:193), IQNTFNFSLKQSKH (SEQ ID NO:194),LLKGTNLSASEQLN (SEQ ID NO:195), KSKEQNVSDDPEST (SEQ ID NO:196),SEELKNYSKQFGQL (SEQ ID NO:197), TYELRNWSNSTCLK (SEQ ID NO:198),LRNWSNSTCLKLAV (SEQ ID NO:199), YYSDQNASSSKESA (SEQ ID NO:200),ISEYWNLTETVAIG (SEQ ID NO:201), KMEDVNCSCEERIR (SEQ ID NO:202),SSLSDNSTRSAQSS (SEQ ID NO:203), PWLQPNTSFWINPL (SEQ ID NO:204),ICKIKNLSGSSEIG (SEQ ID NO:205), QGVGENLTDPSVIK (SEQ ID NO:206), and/orSPERINSTFGLEIK (SEQ ID NO:207). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these TRP-PLIK2c asparagine glycosylation site polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

[0336] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:5 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5806 ofSEQ ID NO:5, b is an integer between 15 to 5820, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:5,and where b is greater than or equal to a+14.

[0337] Features of the Polypeptide Encoded by Gene No:4

[0338] The polypeptide of this gene provided as SEQ ID NO:8 (FIGS.4A-G), encoded by the polynucleotide sequence according to SEQ ID NO:7(FIGS. 4A-G), and/or encoded by the polynucleotide contained within thedeposited clone, TRP-PLIK2d, has significant homology at the nucleotideand amino acid level to the human channel-kinase 1 protein, also knownas the human CHAK1 or TRP-PLIKB 1 protein (CHAKi; Genbank Accession No.gilAF346629; SEQ ID NO:9) ; and the human melastatin 1 protein(Melastatin 1; Genbank Accession No. gil3243075; SEQ ID NO:260). Analignment of the TRP-PLIK2d polypeptide with this protein is provided inFIGS. 5A-F.

[0339] The TRP-PLIK2d polypeptide was determined to share 57.9% identityand 65.7% similarity with the human CHAK1 or TRP-PLIKB1 protein (CHAK1;Genbank Accession No. gilAF346629; SEQ ID NO:9); and was determined toshare 48.0% identity and 58.4% similarity with the human melastatin 1protein (Melastatinl; Genbank Accession No. gil3243075; SEQ ID NO:260)as shown in FIG. 9.

[0340] The CHAK1 protein is believed to represent a member of a newclass of protein kianses referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain.

[0341] The melastatin 1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin 1 could represent a noveltherapeutic in the treatment of melanoa and potentially other cancers.

[0342] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the CHAK1 and melastatin 1 proteins, in addition to,other transient receptor potential channel family members referencedelsewhere herein or otherwise known in the art.

[0343] The TRP-PLIK2d (SEQ ID NO:8) polypeptide represents a novelsplice variant form of the TRP-PLIK2 (SEQ ID NO:2) polypeptide of thepresent invention.

[0344] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theTRP-PLIK2d polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 4A-G. The transmembrane domains arelocated from about amino acid 740 to about amino acid 757 (TM1), fromabout amino acid 834 to about amino acid 845 (TM2), from about aminoacid 861 to about amino acid 880 (TM3), from about amino acid 892 toabout amino acid 909 (TM4), from about amino acid 925 to about aminoacid 946 (TM5), and/or from about amino acid 996 to about amino acid1026 (TM6) of SEQ ID NO:8. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0345] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention: LKIIISILPPTILTLEF(SEQ ID NO:208), IVKFWFYTICIS (SEQ ID NO:209), YWNLTETVAIGLFSAGFVLR (SEQID NO:210), LIYCIDIIFWFSRLLDFF (SEQ ID NO:211), MTANMFYIVIIMAIVLLSFGVA(SEQ ID NO:212), and/or FLTPFLQAVYLFVQYIIMVNLLIAFFNNVYL (SEQ ID NO:213).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of the TRP-PLIK2dtransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0346] In preferred embodiments, the present invention encompasses theuse of N-terminal deletions, C-terminal deletions, or any combination ofN-terminal and C-terminal deletions of any one or more of the TRP-PLIK2dTM1 thru TM6 transmembrane domain polypeptides as antigenic and/orimmunogenic epitopes.

[0347] In preferred embodiments, the present invention also encompassesthe use of N-terminal deletions, C-terminal deletions, or anycombination of N-terminal and C-terminal deletions of any one or more ofthe amino acids intervening (i.e., ion channel extracellular orintracellular loops) the TRP-PLIK2d TM1 thru TM6 transmembrane domainpolypeptides as antigenic and/or immunogenic epitopes.

[0348] The TRP-PLIK2d polypeptide was determined to comprise severalconserved cysteines, at amino acid 21, 34, 38, 41, 47, 49, 311, 367,637, 702, 719, 895. 985, 991, 1071, 1105, 1779, 1818, 1920, 1923, and1924 of SEQ ID No:8 (FIGS. 4A-G). Conservation of cysteines at key aminoacid residues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0349] In confirmation of the TRP-PLIK2d representing a member of thetransient receptor channel family, the TRP-PLIK2d polypeptide wasdetermined to comprise a predicted TRP domain (LWKYNR) located fromabout amino acid 1035 to about amino acid 1040 of SEQ ID NO:8. In thiscontext, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of theabove referenced polypeptide.

[0350] In further confirmation of the TRP-PLIK2d representing a memberof the transient receptor channel family, the TRP-PLIK2d polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 904 to about amino acid 959 of SEQ ID NO:8.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0351] The TRP-PLIK2d polypeptide was determined to comprise a predictednucleotide binding domain located from about amino acid 1902 to aboutamino acid 1907 of SEQ ID NO:8. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0352] In addition, the TRP-PLIK2d polypeptide was determined tocomprise a predicted zinc finger domain located at about amino acid 1917to about amino acid 1927 of SEQ ID NO:8. In this context, the term“about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids beyond the N-Terminus and/or C-terminus of the above referencedpolypeptide.

[0353] TRP-PLIK2d polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofTRP-PLIK2d by identifying mutations in the TRP-PLIK2d gene usingTRP-PLIK2d sequences as probes or by determining TRP-PLIK2d protein ormRNA expression levels. TRP-PLIK2d polypeptides will be useful inscreens for compounds that affect the activity of the protein.TRP-PLIK2d peptides can also be used for the generation of specificantibodies and as bait in yeast two hybrid screens to find proteins thespecifically interact with TRP-PLIK2d.

[0354] Expression profiling designed to measure the steady state mRNAlevels encoding the TRP-PLIK2 polypeptide showed predominately highexpression levels in bone marrow, kidney, and testis. The TRP-PLIK2polypeptide was also significantly expressioned in liver, and to alesser extent, in small intestine, spinal cord, prostate, uterus, lung,lymph node, stomach, heart, brain, thymus, and pancrease (as shown inFIG. 7). The tissue expression of TRP-PLIK2d may follow the same patternas for the TRP-PLIK2 polypeptide of the present invention.

[0355] Expanded analysis of TRP-PLIK2 expression levels by TaqMan™quantitative PCR (see FIG. 12) confirmed that the TRP-PLIK2 polypeptideis expressed in kidney, colon, and testis (FIG. 7). TRP-PLIK2 mRNA wasexpressed predominately in the lower gastrointestinal tract,specifically the ileum, the rectum, the colon, the jejunum, and to alesser extent in the duodenum and stomach. Significant expression wasobserved in the kidney, particularly in the cortex, followed by themedulla, and to a lesser extent in the testis, pelvis, and bone marrow(mononuclear cells).

[0356] Furthermore, an expanded analysis of TRP-PLIK2 expression levelsin various tumor and normal tissues by TaqMan™ quantitative PCR (seeFIG. 14) showed TRP-PLIK2 mRNA was differentially expressed to thegreatest extent in prostate tumor tissue relative to normal prostatetissue (approximately 20 fold difference). Significant differentalexpression was also observed in the testicular tumor tissue relative tonormal testicular tissue.

[0357] In preferred embodiments, TRP-PLIK2d polynucleotides andpolypeptides, including fragments thereof, are useful for treating,diagnosing, and/or ameliorating proliferative disorders, cancers,ischemia-reperfusion injury, heart failure, immuno compromisedconditions, HIV infection, and renal diseases.

[0358] Moreover, TRP-PLIK2d polynucleotides and polypeptides, includingfragments thereof, are useful for increasing NF-kB activity, increasingapoptotic events, and/or decreasing IkBa expression or activity levels.

[0359] In preferred embodiments, antagonists directed against TRP-PLIK2dare useful for treating, diagnosing, and/or ameliorating autoimmunedisorders, disorders related to hyper immune activity, inflammatoryconditions, disorders related to aberrant acute phase responses,hypercongenital conditions, birth defects, necrotic lesions, wounds,organ transplant rejection, conditions related to organ transplantrejection, disorders related to aberrant signal transduction,proliferating disorders, cancers, HIV, and HIV propagation in cellsinfected with other viruses.

[0360] Moreover, antagonists directed against TRP-PLIK2d are useful fordecreasing NF-kB activity, decreasing apoptotic events, and/orincreasing IkBa expression or activity levels.

[0361] In preferred embodiments, agonists directed against TRP-PLIK2dare useful for treating, diagnosing, and/or ameliorating autoimmunediorders, disorders related to hyper immune activity, hypercongenitalconditions, birth defects, necrotic lesions, wounds, disorders relatedto aberrant signal transduction, immuno compromised conditions, HIVinfection, proliferating disorders, and/or cancers.

[0362] Moreover, agonists directed against TRP-PLIK2d are useful forincreasing NF-kB activity, increasing apoptotic events, and/ordecreasing IkBa expression or activity levels.

[0363] The strong homology to transient receptor potential channels(TRP), combined with the predominate localized expression of theTRP-PLIK2 polypeptide in the lower gastrointestinal tract, specificallythe ileum, the rectum, the colon, the jejunum, and to a lesser extent inthe duodenum and stomach, suggests the TRP-PLIK2d polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing gastrointesinal diseases and/or disorders, which include, butare not limited to, ulcers, irritable bowel syndrome, inflammatory boweldisease, diarrhea, traveler's diarrhea, drug-related diarrhea polyps,absorption disorders, constipation, diverticulitis, vascular disease ofthe intestines, intestinal obstruction, intestinal infections,ulcerative colitis, Shigellosis, cholera, Crohn's Disease, amebiasis,enteric fever, Whipple's Disease, peritonitis, intrabdominal abcesses,hereditary hemochromatosis, gastroenteritis, viral gastroenteritis, foodpoisoning, mesenteric ischemia, mesenteric infarction, in addition to,metabolic diseases and/or disorders.

[0364] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosingsusceptibility to the following, non-limiting, gastrointestinalinfections: Salmonella infection, E. coli infection, E.coli 0157:H7infection, Shiga Toxin-producing E. coli infection, Campylobacterinfection (e.g., Campylobacter fetus, Campylobacter upsaliensis,Campylobacter hyointestinalis, Campylobacter lari, Campylobacter jejuni,Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum,Campylobacter rectus, Campylobacter curvus, Campylobacter sputorum,etc.), Heliobacter infection (e.g., Heliobacter cinaedi, Heliobacterfennelliae, etc.) Yersinia enterocolitica infection, Vibrio sp.Infection (e.g., Vibrio mimicus, Vibrio parahaemolyticus, Vibriofluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio vulnificus, Vibrioalginolyticus, Vibrio metschnikovii, Vibrio damsela, Vibriocincinnatiensis, etc.) Aeromonas infection (e.g., Aeromonas hydrophila,Aeromonas sobira, Aeromonas caviae, etc.), Plesiomonas shigelliodesinfection, Giardia infection (e.g., Giardia lamblia, etc.),Cryptosporidium infection, Listeria infection, Entamoeba histolyticainfection, Rotavirus infection, Norwalk virus infection, Clostridiumdifficile infection, Clostriudium perfringens infection, Staphylococcusinfection, Bacillus infection, in addition to any other gastrointestinaldisease and/or disorder implicated by the causative agents listed aboveor elsewhere herein.

[0365] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of the TRP-PLIK2polypeptide in kidney tissue suggests the TRP-PLIK2d polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention ,slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0366] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trpl2 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H.,Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0367] Thus, the TRP-PLIK2d polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein.

[0368] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of the TRP-PLIK2polypeptide in bone marrow tissue suggests the TRP-PLIK2dpolynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing immune diseases and/or disorders.Representative uses are described in the “Immune Activity”,“Chemotaxis”, and “Infectious Disease” sections below, and elsewhereherein. Briefly, the strong expression in immune tissue indicates a rolein regulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.

[0369] The TRP-PLIK2d polypeptide may also be useful as a preventativeagent for immunological disorders including arthritis, asthma,immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis,granulomatous disease, inflammatory bowel disease, sepsis, acne,neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cellmediated cytotoxicity; immune reactions to transplanted organs andtissues, such as host-versus-graft and graft-versus-host diseases, orautoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, andscleroderma. The TRP-PLIK2 polypeptide may be useful for modulatingcytokine production, antigen presentation, or other processes, such asfor boosting immune responses, etc.

[0370] Moreover, the protein may represent a factor that influences thedifferentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Furthermore, the protein may alsobe used to determine biological activity, raise antibodies, astissuemarkers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0371] Significantly, TRP-PLIK2d is believed to represent the first TRPfamily member expressed in bone marrow tissue.

[0372] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression of TRP-PLIK intestis tissue emphasizes the potential utility for TRP-PLIK2dpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0373] In preferred embodiments, TRP-PLIK2d polynucleotides andpolypeptides including agonists, antagonists, and/or fragments thereof,have uses which include treating, diagnosing, prognosing, and/orpreventing the following, non-limiting, diseases or disorders of thetestis: spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The TRP-PLIK2dpolynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0374] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for TRP-PLIK2d polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kalimansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0375] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0376] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I.,Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Birnbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Nati, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0377] Thus, the TRP-PLIK2d polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein.

[0378] The predominate differential expression of TRP-PLIK2 in prostatetumor relative to normal prostate tissue strongly suggests TRP-PLIK2dpolynucleotides and polypeptides including agonists, antagonists, and/orfragments thereof, have uses which include treating, diagnosing,prognosing, and/or preventing prostate cancers and/or proliferativeconditions.

[0379] Alternatively, the tissue distribution of TRP-PLIK2 in liverindicates the protein product of the TRP-PLIK2d clone would be usefulfor the detection and treatment of liver disorders and cancers.Representative uses are described in the “Hyperproliferative Disorders”,“Infectious Disease”, and “Binding Activity” sections below, andelsewhere herein. Briefly, the protein can be used for the detection,treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis,liver metabolic diseases and conditions that are attributable to thedifferentiation of hepatocyte progenitor cells, cirrhosis, hepaticcysts, pyrogenic abscess, amebic abcess, hydatid cyst,cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma,hepatocellulae carcinoma, cholangiocarcinoma, angiosarcoma, andgranulomatous liver disease.

[0380] Moreover, polynucleotides and polypeptides, including fragmentsand/or antagonists thereof, have uses which include, directly orindirectly, treating, preventing, diagnosing, and/or prognosing thefollowing, non-limiting, hepatic infections: liver disease caused bysepsis infection, liver disease caused by bacteremia, liver diseasecaused by Pneomococcal pneumonia infection, liver disease caused byToxic shock syndrome, liver disease caused by Listeriosis, liver diseasecaused by Legionnaries' disease, liver disease caused by Brucellosisinfection, liver disease caused by Neisseria gonorrhoeae infection,liver disease caused by Yersinia infection, liver disease caused bySalmonellosis, liver disease caused by Nocardiosis, liver disease causedby Spirochete infection, liver disease caused by Treponema palliduminfection, liver disease caused by Brrelia burgdorferi infection, liverdisease caused by Leptospirosis, liver disease caused by Coxiellaburnetii infection, liver disease caused by Rickettsia richettsiiinfection, liver disease caused by Chlamydia trachomatis infection,liver disease caused by Chlamydia psittaci infection, in addition to anyother hepatic disease and/or disorder implicated by the causative agentslisted above or elsewhere herein.

[0381] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0382] In preferred embodiments, TRP-PLIK2d polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0383] In more preferred embodiments, TRP-PLIK2d polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0384] TRP-PLIK2d polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since TRP-PLIK2 is dominantly expressed in bone marrow, the TRP-PLIK2dsplice variant may play an important role in regulating cytosolic Ca²⁺in immune system.

[0385] The TRP-PLIK2 gene maps to chromosome 9q21.2-22.1. This region islinked to amyotrophic lateral sclerosis with frontotemporal dementia,early-onset pulverulent cataract, infantile nephronophthisis,hypomagnesemia with secondary hypocalcemia and familial hemophagocyticlymphohistiocytosis. Therefore, agonists and/or antagonists of the novelTRP-PLIK2d splice variant can be used to treat diseases includingvarious forms of neuronal degeneration, neurogenic inflammation,allergy, immunodeficiency/excessive immune activation, visual defects,hearing disorder, pain, cancer, hypertension and other cardiovasculardiseases. In addition, the therapeutics may be useful in the treatmentof diseases associated with disturbances in Ca²⁺ homeostasis includingosteoporosis, hypercalciuric stone disease, and chronic renal failure.

[0386] In addition, TRP-PLIK2d polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ senstive proteins, the activation of Ca++ senstivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0387] The TRP-PLIK2d polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian bone marrow, kidney, testis, liver, small intestine, spinalcord, prostate, uterus, lung, lymph node, stomach, heart, brain, thymus,and pancreas, preferably human. TRP-PLIK2d polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, may be useful in diagnosing, treating, prognosing,and/or preventing immune, hematopoietic, renal, reproductive, hepatic,and/or proliferative diseases or disorders, particularly of the immunesystem.

[0388] In addition, antagonists of the TRP-PLIK2d polynucleotides andpolypeptides may have uses that include diagnosing, treating,prognosing, and/or preventing diseases or disorders related to transientreceptor potential channel activity, which may include immune,hematopoietic, renal, reproductive, hepatic, and/or proliferativediseases or disorders.

[0389] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those from CHAK1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the TRP-PLIK2dpolynucleotides, in addition to, other clones of the present invention,may be arrayed on microchips for expression profiling. Depending onwhich polynucleotide probe is used to hybridize to the slides, a changein expression of a specific gene may provide additional insight into thefunction of this gene based upon the conditions being studied. Forexample, an observed increase or decrease in expression levels when thepolynucleotide probe used comes from tissue that has been treated withknown immunoglobulin inhibitors, which include, but are not limited tothe drugs listed herein or otherwise known in the art, might indicate afunction in modulating immunoglobulin function, for example. In the caseof TRP-PLIK2d, bone marrow, kidney, testis, liver, small intestine,spinal cord, prostate, uterus, lung, lymph node, stomach, heart, brain,thymus, and/or pancrease, should be used to extract RNA to prepare theprobe.

[0390] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the TRP-PLIK2d gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:7 (FIGS. 4A-G).

[0391] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theTRP-PLIK2d, transforming yeast deficient in transient receptor potentialchannel activity with TRP-PLIK2d and assessing their ability to growwould provide convincing evidence the TRP-PLIK2d polypeptide hastransient receptor potential channel activity. Additional assayconditions and methods that may be used in assessing the function of thepolynucletides and polypeptides of the present invention are known inthe art, some of which are disclosed elsewhere herein.

[0392] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0393] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a bone marrow,kidney, testis, liver, small intestine, spinal cord, prostate, uterus,lung, lymph node, stomach, heart, brain, thymus, and/orpancrease-specific promoter), or it can be expressed at a specified timeof development using an inducible and/or a developmentally regulatedpromoter.

[0394] In the case of TRP-PLIK2d transgenic mice or rats, if nophenotype is apparent in normal growth conditions, observing theorganism under diseased conditions (immune, hematopoietic, renal,reproductive, hepatic, or proliferative disorders, etc.) may lead tounderstanding the function of the gene. Therefore, the application ofantisense and/or sense methodology to the creation of transgenic mice orrats to refine the biological function of the polypeptide is encompassedby the present invention.

[0395] In preferred embodiments, the following N-terminal TRP-PLIK2ddeletion polypeptides are encompassed by the present invention:M1-L1974, 12-L1974, 13-L1974, L4-L1974, S5-L1974, K6-L1974, S7-L1974,Q8-L1974, K9-L1974, S10-L1974, WII-L1974, 112-L1974, K13-L1974,G14-L1974, V15-L1974, F16-L1974, D17-L1974, K18-L1974, R19-L1974,E20-L1974, C21-L1974, S22-L1974, T23-L1974, 124-L1974, 125-L1974,P26-L1974, S27-L1974, S28-L1974, K29-L1974, N30-L1974, P31-L1974,H32-L1974, R33-L1974, C34-L1974, T35-L1974, P36-L1974, V37-L1974,C38-L1974, Q39-L1974, V40-L1974, C41-L1974, Q42-L1974, N43-L1974,L44-L1974, 145-L1974, R46-L1974, C47-L1974, Y48-L1974, C49-L1974,G50-L1974, R51-L1974, L52-L1974, 153-L1974, G54-L1974, D55-L1974,H56-L1974, A57-L1974, G58-L1974, 159-L1974, D60-L1974, Y61-L1974,S62-L1974, W63-L1974, T64-L1974, 165-L1974, S66-L1974, A67-L1974,A68-L1974, K69-L1974, G70-L1974, K71-L1974, E72-L1974, S73-L1974,E74-L1974, Q75-L1974, W76-L1974, S77-L1974, V78-L1974, E79-L1974,K80-L1974, H81-L1974, T82-L1974, T83-L1974, K84-L1974, S85-L1974,P86-L1974, T87-L1974, D88-L1974, T89-L1974, F90-L1974, G91-L1974,T92-L1974, 193-L1974, N94-L1974, F95-L1974, Q96-L1974, D97-L1974,G98-L1974, E99-L1974, H100-L1974, T101-L1974, H102-L1974, H103-L1974,A104-L1974, K105-L1974, Y106-L1974, 1107-L1974, R108-L1974, T109-L1974,S110-L1974, Y111-L1974, D112-L1974, T113-L1974, K114-L1974, L115-L1974,D116-L1974, H117-L1974, L118-L1974, LIl9-L1974, H120-L1974, L121-L1974,M122-L1974, L123-L1974, K124-L1974, E125-L1974, W126-L1974, K127-L1974,M128-L1974, E129-L1974, L130-L1974, P131-L1974, K132-L1974, L133-L1974,V134-L1974, 1135-L1974, S136-L1974, V137-L1974, H138-L1974, G139-L1974,G140-L1974, 1141-L1974, Q142-L1974, N143-L1974, F144-L1974, T145-L1974,M146-L1974, P147-L1974, S148-L1974, K149-L1974, F150-L1974, K151-L1974,E152-L1974, 1153-L1974, F154-L1974, S155-L1974, Q156-L1974, G157-L1974,L158-L1974, V159-L1974, K160-L1974, A161-L1974, A162-L1974, E163-L1974,T164-L1974, T165-L1974, G166-L1974, A167-L1974, W168-L1974, 1169-L1974,1170-L1974, T171-L1974, E172-L1974, G173-L1974, 1174-L1974, N175-L1974,T176-L1974, G177-L1974, V178-L1974, S179-L1974, K180-L1974, H181-L1974,V182-L1974, G183-L1974, D184-L1974, A185-L1974, L186-L1974, K187-L1974,S188-L1974, H189-L1974, S190-L1974, S191-L1974, H192-L1974, S193-L1974,L194-L1974, R195-L1974, K196-L1974, I197-L1974, W198-L1974, T199-L1974,V200-L1974, G201-L1974, 1202-L1974, P203-L1974, P204-L1974, W205-L1974,G206-L1974, V207-L1974, 1208-L1974, E209-L1974, N210-L1974, Q211-L1974,R212-L1974, D213-L1974, L214-L1974, 1215-L1974, G216-L1974, K217-L1974,D218-L1974, V219-L1974, V220-L1974, C221-L1974, L222-L1974, Y223-L1974,Q224-L1974, T225-L1974, L226-L1974, D227-L1974, N228-L1974, P229-L1974,L230-L1974, S231-L1974, K232-L1974, L233-L1974, T234-L1974, T235-L1974,L236-L1974, N237-L1974, S238-L1974, M239-L1974, H240-L1974, S241-L1974,H242-L1974, F243-L1974, 1244-L1974, L245-L1974, S246-L1974, D247-L1974,D248-L1974, G249-L1974, T250-L1974, V251-L1974, G252-L1974, K253-L1974,Y254-L1974, G255-L1974, N256-L1974, E257-L1974, M258-L1974, K259-L1974,L260-L1974, R261-L1974, R262-L1974, N263-L1974, L264-L1974, E265-L1974,K266-L1974, Y267-L1974, L268-L1974, S269-L1974, L270-L1974, Q271-L1974,K272-L1974, 1273-L1974, H274-L1974, C275-L1974, R276-L1974, S277-L1974,R278-L1974, Q279-L1974, G280-L1974, V281-L1974, P282-L1974, V283-L1974,V284-L1974, G285-L1974, L286-L1974, V287-L1974, V288-L1974, E289-L1974,G290-L1974, G291-L1974, P292-L1974, N293-L1974, V294-L1974, 1295-L1974,L296-L1974, S297-L1974, V298-L1974, W299-L1974, E300-L1974, T301-L1974,V302-L1974, K303-L1974, D304-L1974, K305-L1974, D306-L1974, P307-L1974,V308-L1974, V309-L1974, V310-L1974, C311-L1974, E312-L1974, G313-L1974,T314-L1974, G315-L1974, R316-L1974, A317-L1974, A318-L1974, D319-L1974,L320-L1974, L321-L1974, A322-L1974, F323-L1974, T324-L1974, H325-L1974,K326-L1974, H327-L1974, L328-L1974, A329-L1974, D330-L1974, E331-L1974,G332-L1974, M333-L1974, L334-L1974, R335-L1974, P336-L1974, Q337-L1974,V338-L1974, K339-L1974, E340-L1974, E341-L1974, 1342-L1974, 1343-L1974,C344-L1974, M345-L1974, 1346-L1974, Q347-L1974, N348-L1974, T349-L1974,F350-L1974, N351-L1974, F352-L1974, S353-L1974, L354-L1974, K355-L1974,Q356-L1974, S357-L1974, K358-L1974, H359-L1974, L360-L1974, F361-L1974,Q362-L1974, 1363-L1974, L364-L1974, M365-L1974, E366-L1974, C367-L1974,M368-L1974, V369-L1974, H370-L1974, R371-L1974, D372-L1974, C373-L1974,1374-L1974, T375-L1974, 1376-L1974, F377-L1974, D378-L1974, A379-L1974,D380-L1974, S381-L1974, E382-L1974, E383-L1974, Q384-L1974, Q385-L1974,D386-L1974, L387-L1974, D388-L1974, L389-L1974, A390-L1974, 1391-L1974,L392-L1974, T393-L1974, A394-L1974, L395-L1974, L396-L1974, K397-L1974,G398-L1974, T399-L1974, N400-L1974, L401-L1974, S402-L1974, A403-L1974,S404-L1974, E405-L1974, Q406-L1974, L407-L1974, N408-L1974, L409-L1974,A410-L1974, M411-L1974, A412-L1974, W413-L1974, D414-L1974, R415-L1974,V416-L1974, D417-L1974, 1418-L1974, A419-L1974, K420-L1974, K421-L1974,H422-L1974, 1423-L1974, L424-L1974, 1425-L1974, Y426-L1974, E427-L1974,Q428-L1974, H429-L1974, W430-L1974, K431-L1974, P432-L1974, D433-L1974,A434-L1974, L435-L1974, E436-L1974, Q437-L1974, A438-L1974, M439-L1974,S440-L1974, D441-L1974, A442-L1974, L443-L1974, V444-L1974, M445-L1974,D446-L1974, R447-L1974, V448-L1974, D449-L1974, F450-L1974, V451-L1974,K452-L1974, L453-L1974, L454-L1974, 1455-L1974, E456-L1974, Y457-L1974,G458-L1974, V459-L1974, N460-L1974, L461-L1974, H462-L1974, R463-L1974,F464-L1974, L465-L1974, T466-L1974, 1467-L1974, P468-L1974, R469-L1974,L470-L1974, E471-L1974, E472-L1974, IA73-L1974, Y474-L1974, N475-L1974,T476-L1974, K477-L1974, Q478-L1974, G479-L1974, P480-L1974, T481-L1974,N482-L1974, T483-L1974, L484-L1974, L485-L1974, H486-L1974, H487-L1974,L488-L1974, V489-L1974, Q490-L1974, D491-L1974, V492-L1974, K493-L1974,Q494-L1974, H495-L1974, T496-L1974, L497-L1974, L498-L1974, S499-L1974,G500-L1974, Y501-L1974, R502-L1974, 1503-L1974, T504-L1974, L505-L1974,1506-L1974, D507-L1974, I508-L1974, G509-L1974, L510-L1974, V511-L1974,V512-L1974, E513-L1974, Y514-L1974, L515-L1974, 1516-L1974, G517-L1974,R518-L1974, A519-L1974, Y520-L1974, R521-L1974, S522-L1974, N523-L1974,Y524-L1974, T525-L1974, R526-L1974, K527-L1974, H528-L1974, F529-L1974,R530-L1974, A531-L1974, L532-L1974, Y533-L1974, N534-L1974, N535-L1974,L536-L1974, Y537-L1974, R538-L1974, K539-L1974, Y540-L1974, K541-L1974,H542-L1974, Q543-L1974, R544-L1974, H545-L1974, S546-L1974, S547-L1974,G548-L1974, N549-L1974, R550-L1974, N551-L1974, E552-L1974, S553-L1974,A554-L1974, E555-L1974, S556-L1974, T557-L1974, L558-L1974, H559-L1974,S560-L1974, Q561-L1974, F562-L1974, 1563-L1974, R564-L1974, T565-L1974,A566-L1974, Q567-L1974, P568-L1974, Y569-L1974, K570-L1974, F571-L1974,K572-L1974, E573-L1974, K574-L1974, S575-L1974, 1576-L1974, V577-L1974,L578-L1974, H579-L1974, K580-L1974, S581-L1974, R582-L1974, K583-L1974,K584-L1974, S585-L1974, K586-L1974, E587-L1974, Q588-L1974, N589-L1974,V590-L1974, S591-L1974, D592-L1974, D593-L1974, P594-L1974, E595-L1974,S596-L1974, T597-L1974, G598-L1974, F599-L1974, L600-L1974, Y601-L1974,P602-L1974, Y603-L1974, N604-L1974, D605-L1974, L606-L1974, L607-L1974,V608-L1974, W609-L1974, A610-L1974, V611-L1974, L612-L1974, M613-L1974,K614-L1974, R615-L1974, Q616-L1974, K617-L1974, M618-L1974, A619-L1974,M620-L1974, F621-L1974, F622-L1974, W623-L1974, Q624-L1974, H625-L1974,G626-L1974, E627-L1974, E628-L1974, A629-L1974, T630-L1974, V631-L1974,K632-L1974, A633-L1974, V634-L1974, 1635-L1974, A636-L1974, C637-L1974,1638-L1974, L639-L1974, Y640-L1974, R641-L1974, A642-L1974, M643-L1974,A644-L1974, H645-L1974, E646-L1974, A647-L1974, K648-L1974, E649-L1974,S650-L1974, H651-L1974, M652-L1974, V653-L1974, D654-L1974, D655-L1974,A656-L1974, S657-L1974, E658-L1974, E659-L1974, L660-L1974, K661-L1974,N662-L1974, Y663-L1974, S664-L1974, K665-L1974, Q666-L1974, F667-L1974,G668-L1974, Q669-L1974, L670-L1974, A671-L1974, L672-L1974, D673-L1974,L674-L1974, L675-L1974, E676-L1974, K677-L1974, A678-L1974, F679-L1974,K680-L1974, Q681-L1974, N682-L1974, E683-L1974, R684-L1974, M685-L1974,A686-L1974, M687-L1974, T688-L1974, L689-L1974, L690-L1974, T691-L1974,Y692-L1974, E693-L1974, L694-L1974, R695-L1974, N696-L1974, W697-L1974,S698-L1974, N699-L1974, S700-L1974, T701-L1974, C702-L1974, L703-L1974,K704-L1974, L705-L1974, A706-L1974, V707-L1974, S708-L1974, G709-L1974,G710-L1974, L711-L1974, R712-L1974, P713-L1974, F714-L1974, V715-L1974,S716-L1974, H717-L1974, T718-L1974, C719-L1974, T720-L1974, Q721-L1974,M722-L1974, L723-L1974, L724-L1974, T725-L1974, D726-L1974, M727-L1974,W728-L1974, M729-L1974, G730-L1974, R731-L1974, L732-L1974, K733-L1974,M734-L1974, R735-L1974, K736-L1974, N737-L1974, S738-L1974, W739-L1974,L740-L1974, K741-L1974, 1742-L1974, 1743-L1974, 1744-L1974, S745-L1974,1746-L1974, 1747-L1974, L748-L1974, P749-L1974, P750-L1974, T751-L1974,1752-L1974, L753-L1974, T754-L1974, L755-L1974, E756-L1974, F757-L1974,K758-L1974, S759-L1974, K760-L1974, A761-L1974, E762-L1974, M763-L1974,S764-L1974, H765-L1974, V766-L1974, P767-L1974, Q768-L1974, S769-L1974,Q770-L1974, D771-L1974, F772-L1974, Q773-L1974, F774-L1974, M775-L1974,W776-L1974, Y777-L1974, Y778-L1974, S779-L1974, D780-L1974, Q781-L1974,N782-L1974, A783-L1974, S784-L1974, S785-L1974, S786-L1974, K787-L1974,E788-L1974, S789-L1974, A790-L1974, S791-L1974, V792-L1974, K793-L1974,E794-L1974, Y795-L1974, D796-L1974, L797-L1974, E798-L1974, R799-L1974,G800-L1974, H801-L1974, D802-L1974, E803-L1974, K804-L1974, L805-L1974,D806-L1974, E807-L1974, N808-L1974, Q809-L1974, H810-L1974, F811-L1974,G812-L1974, L813-L1974, E814-L1974, S815-L1974, G816-L1974, H817-L1974,Q818-L1974, H819-L1974, L820-L1974, P821-L1974, W822-L1974, T823-L1974,R824-L1974, K825-L1974, V826-L1974, Y827-L1974, E828-L1974, F829-L1974,Y830-L1974, S831-L1974, A832-L1974, P833-L1974, 1834-L1974, V835-L1974,K836-L1974, F837-L1974, W838-L1974, F839-L1974, Y840-L1974, and/orT841-L1974 of SEQ ID NO:8. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal TRP-PLIK2d deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0396] In preferred embodiments, the following C-terninal TRP-PLIK2ddeletion polypeptides are encompassed by the present invention:M1-L1974, M1-Q1973, M1-M1972, M1-D1971, M1-D1970, M1-E1969, M1-P1968,M1-S1967, M1-N1966, M1-R1965, M1-G1964, M1-T1963, M1-E1962, M1-R1961,M1-A1960, M1-P1959, M1-P1958, M1-E1957, M1-E1956, M1-A1955, M1-S1954,M1-E1953, M1-I1952, M1-K1951, M1-I1950, M1-E1949, M1-L1948, M1-G1947,M1-F1946, M1-T1945, M1-S1944, M1-N1943, M1-I1942, M1-R1941, M1-E1940,M1-P1939, M1-S1938, M1-Y1937, M1-D1936, M1-N1935, M1-R1934, M1-K1933,M1-L1932, M1-D1931, M1-P1930, M1-L1929, M1-K1928, M1-L1927, M1-K1926,M1-R1925, M1-C1924, M1-C1923, M1-S1922, M1-N1921, M1-C1920, M1-H1919,M1-H1918, M1-K1917, M1-A1916, M1-I1915, M1-F1914, M1-N1913, M1-R1912,M1-I1911, M1-A1910, M1-D1909, M1-E1908, M1-G1907, M1-L1906, M1-N1905,M1-A1904, M1-P1903, M1-G1902, M1-F1901, M1-V1900, M1-M1899, M1-G1898,M1-R1897, M1-S1896, M1-Q1895, M1-K1894, M1-V1893, M1-E1892, M1-P1891,M1-K1890, M1-I1889, M1-V1888, M1-S1887, M1-P1886, M1-D1885, M1-T1884,M1-L1883, M1-N1882, M1-E1881, M1-G1880, M1-V1879, M1-G1878, M1-Q1877,M1-L1876, M1-D1875, M1-L1874, M1-V1873, M1-L1872, M1-L1871, M1-E1870,M1-G1869, M1-R1868, M1-T1867, M1-Y1866, M1-E1865, M1-Y1864, M1-T1863,M1-W1862, M1-H1861, M1-S1860, M1-F1859, M1-A1858, M1-L1857, M1-M1856,M1-L1855, M1-E1854, M1-E1853, M1-L1852, M1-T1851, M1-N1850, M1-T1849,M1-P1848, M1-T1847, M1-I1846, M1-E1845, M1-D1844, M1-G1843, M1-N1842,M1-N1841, M1-N1840, M1-N1839, M1-Y1838, M1-K1837, M1-R1836, M1-F1835,M1-E1834, M1-G1833, M1-T1832, M1-M1831, M1-Y1830, M1-K1829, M1-E1828,M1-I1827, M1-T1826, M1-L1825, M1-W1824, M1-Q1823, M1-N1822, M1-A1821,M1-S1820, M1-H1819, M1-C1818, M1-Y1817, M1-11816, M1-L1815, M1-F1814,M1-V1813, M1-E1812, M1-L1811, M1-F1810, M1-R1809, M1-P1808, M1-T1807,M1-Y1806, M1-P1805, M1-I1804, M1-T1803, M1-Q1802, M1-P1801, M1-K1800,M1-V1799, M1-Q1798, M1-N1797, M1-F1796, M1-T1795, M1-Y1794, M1-I1793,M1-L1792, M1-K1791, M1-Q1790, M1-A1789, M1-A1788, M1-R1787, M1-Q1786,M1-Q1785, M1-Q1784, M1-I1783, M1-E1782, M1-R1781, M1-L1780, M1-C1779,M1-L1778, M1-H1777, M1-L1776, M1-V1775, M1-T1774, M1-S1773, M1-E1772,M1-Q1771, M1-F1770, M1-I1769, M1-K1768, M1-H1767, M1-W1766, M1-T1765,M1-R1764, M1-V1763, M1-V1762, M1-E1761, M1-Pl760, M1-L1759, M1-F1758,M1-S1757, M1-K1756, M1-V1755, M1-I1754, M1-F1753, M1-V1752, M1-Q1751,M1-G1750, M1-P1749, M1-K1748, M1-L1747, M1-I1746, M1-D1745, M1-D1744,M1-E1743, M1-S1742, M1-W1741, M1-T1740, M1-S1739, M1-V1738, M1-V1737,M1-R1736, M1-M1735, M1-A1734, M1-K1733, M1-R1732, M1-L1731, M1-G1730,M1-G1729, M1-D1728, M1-M1727, M1-E1726, M1-E1725, M1-R1724, M1-S1723,M1-L1722, M1-V1721, M1-Q1720, M1-I1719, M1-M1718, M1-A1717, M1-A1716,M1-R1715, M1-G1714, M1-R1713, M1-Q1712, M1-S1711, M1-W1710, M1-S1709,M1-S1708, M1-M1707, M1-S1706, M1-K1705, M1-D1704, M1-L1703, M1-N1702,M1-L1701, M1-P1700, M1-S1699, M1-S1698, M1-E1697, M1-E1696, M1-L1695,M1-R1694, M1-Y1693, M1-V1692, M1-T1691, M1-I1690, M1-E1689, M1-E1688,M1-G1687, M1-A1686, M1-F1685, M1-L1684, M1-Q1683, M1-V1682, M1-P1681,M1-T1680, M1-F1679, M1-P1678, M1-I1677, M1-T1676, M1-Q1675, M1-S1674,M1-L1673, M1-R1672, M1-M1671, M1-L1670, M1-N1669, M1-N1668, M1-R1667,M1-E1666, M1-I1665, M1-A1664, M1-S1663, M1-Y1662, M1-H1661, M1-H1660,M1-H1659, M1-P1658, M1-E1657, M1-Q1656, M1-P1655, M1-S1654, M1-K1653,M1-L1652, M1-S1651, M1-A1650, M1-S1649, M1-I1648, M1-K1647, M1-D1646,M1-V1645, M1-G1644, M1-I1643, M1-S1642, M1-S1641, M1-K1640, M1-L1639,M1-L1638, M1-S1637, M1-N1636, M1-R1635, M1-N1634, M1-L1633, M1-N1632,M1-T1631, M1-S1630, M1-R1629, M1-S1628, M1-N1627, M1-W1626, M1-L1625,M1-S1624, M1-N1623, M1-K1622, M1-S1621, M1-L1620, M1-D1619, M1-E1618,M1-Q1617, M1-S1616, M1-Q1615, M1-K1614, M1-L1613, M1-Y1612, M1-D1611,M1-S1610, M1-I1609, M1-Q1608, M1-I1607, M1-A1606, M1-C1605, M1-Q1604,M1-G1603, M1-I1602, M1-E1601, M1-K1600, M1-T1599, M1-K1598, M1-M1597,M1-K1596, M1-Q1595, M1-H1594, M1-I1593, M1-Y1592, M1-P1591, M1-E1590,M1-V1589, M1-G1588, M1-T1587, M1-H1586, M1-S1585, M1-F1584, M1-K1583,M1-S1582, M1-V1581, M1-T1580, M1-F1579, M1-W1578, M1-N1577, M1-K1576,M1-S1575, M1-Y1574, M1-E1573, M1-E1572, M1-E1571, M1-S1570, M1-I1569,M1-S1568, M1-N1567, M1-E1566, M1-G1565, M1-P1564, M1-E1563, M1-P1562,M1-N1561, M1-L1560, M1-Q1559, M1-D1558, M1-S1557, M1-Q1556, M1-S1555,M1-C1554, M1-A1553, M1-N1552, M1-V1551, M1-T1550, M1-I1549, M1-I1548,M1-P1547, M1-V1546, M1-Q1545, M1-L1544, M1-G1543, M1-Q1542, M1-T1541,M1-N1540, M1-K1539, M1-K1538, M1-K1537, M1-K1536, M1-S1535, M1-L1534,M1-R1533, M1-R1532, M1-D1531, M1-K1530, M1-T1529, M1-L1528, M1-M1527,M1-K1526, M1-A1525, M1-K1524, M1-V1523, M1-W1522, M1-A1521, M1-G1520,M1-Q1519, M1-G1518, M1-I1517, M1-E1516, M1-S1515, M1-S1514, M1-G1513,M1-S1512, M1-L1511, M1-N1510, M1-K1509, M1-I1508, M1-K1507, M1-C1506,M1-I1505, M1-K1504, M1-M1503, M1-L1502, M1-K1501, M1-E1500, M1-E1499,M1-K1498, M1-H1497, M1-F1496, M1-R1495, M1-F1494, M1-S1493, M1-H1492,M1-S1491, M1-R1490, M1-A1489, M1-F1488, M1-P1487, M1-R1486, M1-Y1485,M1-R1484, M1-R1483, M1-L1482, M1-P1481, M1-N1480, M1-I1479, M1-W1478,M1-F1477, M1-S1476, M1-T1475, M1-N1474, M1-P1473, M1-Q1472, M1-L1471,M1-W1470, M1-P1469, M1-G1468, M1-V1467, M1-E1466, M1-S1465, M1-C1464,M1-E1463, M1-S1462, M1-S1461, M1-Q1460, M1-A1459, M1-S1458, M1-R1457,M1-T1456, M1-S1455, M1-N1454, M1-D1453, M1-S1452, M1-L1451, M1-S1450,M1-S1449, M1-D1448, M1-Q1447, M1-A1446, M1-Q1445, M1-K1444, M1-Q1443,M1-H1442, M1-Q1441, M1-E1440, M1-S1439, M1-R1438, M1-S1437, M1-S1436,M1-D1435, M1-S1434, M1-D1433, M1-C1432, M1-T1431, M1-S1430, M1-P1429,M1-L1428, M1-C1427, M1-T1426, M1-Q1425, M1-W1424, M1-K1423, M1-K1422,M1-K1421, M1-I1420, M1-S1419, M1-F1418, M1-V1417, M1-G1416, M1-T1415,M1-E1414, M1-D1413, M1-G1412, M1-E1411, M1-S1410, M1-F1409, M1-A1408,M1-W1407, M1-N1406, M1-V1405, M1-Y1404, M1-G1403, M1-G1402, M1-G1401,M1-T1400, M1-Q1399, M1-M1398, M1-I1397, M1-K1396, M1-A1395, M1-Q1394,M1-S1393, M1-L1392, M1-P1391, M1-S1390, M1-S1389, M1-M1388, M1-T1387,M1-M1386, M1-P1385, M1-E1384, M1-P1383, M1-T1382, M1-C1381, M1-S1380,M1-L1379, M1-T1378, M1-P1377, M1-L1376, M1-V1375, M1-Q1374, M1-E1373,M1-A1372, M1-K1371, M1-D1370, M1-Q1369, M1-G1368, M1-D1367, M1-L1366,M1-L1365, M1-H1364, M1-A1363, M1-I1362, M1-P1361, M1-E1360, M1-H1359,M1-K1358, M1-E1357, M1-K1356, M1-P1355, M1-E1354, M1-D1353, M1-V1352,M1-S1351, M1-A1350, M1-W1349, M1-D1348, M1-S1347, M1-V1346, M1-V1345,M1-P1344, M1-T1343, M1-Q1342, M1-G1341, M1-T1340, M1-L1339, M1-H1338,M1-V1337, M1-L1336, M1-V1335, M1-E1334, M1-T1333, M1-Q1332, M1-I1331,M1-D1330, M1-Q1329, M1-E1328, M1-T1327, M1-A1326, M1-L1325, M1-V1324,M1-D1323, M1-P1322, M1-V1321, M1-S1320, M1-P1319, M1-R1318, M1-S1317,M1-L1316, M1-P1315, M1-L1314, M1-V1313, M1-T1312, M1-E1311, M1-A1310,M1-S1309, M1-F1308, M1-P1307, M1-V1306, M1-R1305, M1-K1304, M1-L1303,M1-N1302, M1-S1301, M1-P1300, M1-V1299, M1-L1298, M1-L1297, M1-F1296,M1-Q1295, M1-G1294, M1-Y1293, M1-K1292, M1-S1291, M1-H1290, M1-A1289,M1-Q1288, M1-R1287, M1-N1286, M1-P1285, M1-S1284, M1-V1283, M1-G1282,M1-S1281, M1-V1280, M1-V1279, M1-I1278, M1-S1277, M1-S1276, M1-Q1275,M1-T1274, M1-E1273, M1-Q1272, M1-R1271, M1-E1270, M1-Q1269, M1-D1268,M1-N1267, M1-R1266, M1-V1265, M1-N1264, M1-T1263, M1-A1262, M1-E1261,M1-R1260, M1-K1259, M1-S1258, M1-N1257, M1-T1256, M1-I1255, M1-E1254,M1-L1253, M1-L1252, M1-A1251, M1-G1250, M1-R1249, M1-Q1248, M1-V1247,M1-R1246, M1-P1245, M1-P1244, M1-H1243, M1-R1242, M1-G1241, M1-G1240,M1-A1239, M1-L1238, M1-S1237, M1-R1236, M1-L1235, M1-L1234, M1-S1233,M1-S1232, M1-P1231, M1-M1230, M1-S1229, M1-Y1228, M1-Y1227, M1-Q1226,M1-Y1225, M1-K1224, M1-K1223, M1-E1222, M1-G1221, M1-A1220, M1-I1219,M1-E1218, M1-M1217, M1-S1216, M1-G1215, M1-L1214, M1-V1213, M1-E1212,M1-A1211, M1-C1210, M1-I1209, M1-V1208, M1-N1207, M1-S1206, M1-W1205,M1-S1204, M1-H1203, M1-P1202, M1-L1201, M1-K1200, M1-K1199, M1-C1198,M1-T1197, M1-S1196, M1-H1195, M1-K1194, M1-R1193, M1-K1192, M1-A1191,M1-L1190, M1-L1189, M1-A1188, M1-E1187, M1-D1186, M1-E1185, M1-Q1184,M1-L1183, M1-T1182, M1-D1181, M1-V1180, M1-A1179, M1-S1178, M1-L1177,M1-V1176, M1-K1175, M1-L1174, M1-T1173, M1-D1172, M1-V1171, M1-T1170,M1-L1169, M1-A1168, M1-S1167, M1-L1166, M1-D1165, M1-Q1164, M1-L1163,M1-H1162, M1-G1161, M1-V1160, M1-Q1159, M1-S1158, M1-D1157, M1-L1156,M1-S1155, M1-L1154, M1-L1153, M1-S1152, M1-D1151, M1-K1150, M1-I1149,M1-F1148, M1-S1147, M1-V1146, M1-K1145, M1-E1144, M1-N1143, M1-M1142,M1-E1141, M1-K1140, M1-L1139, M1-Q1138, M1-F1137, M1-Y1136, M1-M1135,M1-E1134, M1-T1133, M1-V1132, M1-R1131, M1-E1130, M1-S1129, M1-T1128,M1-V1127, M1-R1126, M1-I1125, M1-R1124, M1-E1123, M1-E1122, M1-C1121,M1-S1120, M1-C1119, M1-N1118, M1-V1117, M1-D1116, M1-E1115, M1-M1114,M1-K1113, M1-E1112, M1-H1111, M1-F1110, M1-Y1109, M1-K1108, M1-E1107,M1-V1106, M1-C1105, M1-Q1104, M1-E1103, M1-E1102, M1-F1101, M1-D1100,M1-H1099, M1-L1098, M1-K1097, M1-K1096, M1-L1095, M1-D1094, M1-E1093,M1-K1092, M1-S1091, M1-L1090, M1-Y1089, M1-L1088, M1-K1087, M1-L1086,M1-G1085, M1-V1084, M1-D1083, M1-G1082, M1-E1081, M1-E1080, M1-Q1079,M1-D1078, M1-H1077, M1-P1076, M1-A1075, M1-R1074, M1-H1073, M1-C1072,M1-C1071, M1-L1070, M1-R1069, M1-R1068, M1-L1067, M1-L1066, M1-L1065,M1-G1064, M1-V1063, M1-H1062, M1-S1061, M1-L1060, M1-L1059, M1-I1058,M1-L1057, M1-P1056, M1-P1055, M1-P1054, M1-L1053, M1-W1052, M1-P1051,M1-K1050, M1-E1049, M1-H1048, M1-Y1047, M1-T1046, M1-M1045, M1-I1044,M1-Y1043, M1-R1042, M1-Y1041, M1-R1040, M1-N1039, M1-Y1038, M1-K1037,M1-W1036, M1-L1035, M1-N1034, M1-NIO33, M1-S1032, M1-I1031, M1-S1030,M1-E1029, M1-M1028, M1-D1027, M1-L1026, M1-Y1025, M1-V1024, M1-N1023,M1-N1022, M1-F1021, M1-F1020, M1-A1019, M1-I1018, M1-L1017, M1-L1016,M1-N1015, M1-V1014, M1-M1013, M1-I1012, M1-I1011, M1-Y1010, M1-Q1009,M1-V1008, M1-F1007, M1-L1006, M1-Y1005, M1-V1004, M1-A1003, M1-Q1002,M1-L1001, M1-F1000, M1-P999, M1-T998, M1-L997, M1-F996, M1-S995,M1-G994, M1-P993, M1-P992, M1-C991, M1-S990, M1-P989, M1-Q988, M1-S987,M1-S986, M1-C985, M1-V984, M1-D983, M1-1982, M1-E981, M1-G980, M1-A979,M1-Y978, M1-V977, M1-E976, M1-G975, M1-Y974, M1-1973, M1-M972, M1-W971,M1-Y970, M1-P969, M1-E968, M1-F967, M1-V966, Ml -1965, M1-D964, M1-R963,M1-A962, M1-L961, M1-S960, M1-W959, M1-S958, M1-P957, M1-P956, M1-E955,M1-K954, M1-P953, M1-S952, M1-L951, M1-1950, M1-A949, M1-K948, M1-R947,M1-A946, M1-V945, M1-G944, M1-F943, M1-S942, M1-L941, M1-L940, M1-V939,M1-1938, M1-A937, M1-M936, M1-1935, M1-1934, M1-V933, M1-1932, M1-Y931,M1-F930, M1-M929, M1-N928, M1-A927, M1-T926, M1-M925, M1-K924, M1-A923,M1-1922, M1-M921, M1-T920, M1-V919, M1-Y918, M1-P917, M1-G916, M1-A915,M1-H914, M1-Q913, M1-N912, M1-V911, M1-A910, M1-F909, M1-F908, M1-D907,M1-L906, M1-L905, M1-R904, M1-S903, M1-F902, M1-W901, M1-F900, M1-1899,M1-1898, M1-D897, M1-1896, M1-C895, M1-Y894, M1-1893, M1-L892, M1-R891,M1-G890, M1-A889, M1-T888, M1-H887, M1-F886, M1-P885, M1-P884, M1-D883,M1-G882, M1-W881, M1-R880, M1-L879, M1-V878, M1-F877, M1-G876, M1-A875,M1-S874, M1-F873, M1-L872, M1-G871, M-1870, M1-A869, M1-V868, M1-T867,M1-E866, M1-T865, M1-L864, M1-N863, M1-W862, M1-Y861, M1-E860, M1-S859,M1-1858, M1-W857, M1-V856, M1-K855, M1-V854, M1-K853, M1-Q852, M1-T851,M1-F850, M1-K849, M1-G848, M1-P847, M1-E846, M1-S845, M1-1844, M1-C843,and/or M1-1842 of SEQ ID NO:8. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal TRP-PLIK2d deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0397] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the TRP-PLIK2d polypeptide (e.g., any combination ofboth N- and C- terminal TRP-PLIK2d polypeptide deletions) of SEQ IDNO:8. For example, internal regions could be defined by the equation:amino acid NX to amino acid CX, wherein NX refers to any N-terminaldeletion polypeptide amino acid of TRP-PLIK2d (SEQ ID NO:8), and whereCX refers to any C-terminal deletion polypeptide amino acid ofTRP-PLIK2d (SEQ ID NO:8). Polynucleotides encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese polypeptides as an immunogenic and/or antigenic epitope asdescribed elsewhere herein.

[0398] The TRP-PLIK2d polypeptides of the present invention weredetermined to comprise several phosphorylation sites based upon theMotif algorithm (Genetics Computer Group, Inc.). The phosphorylation ofsuch sites may regulate some biological activity of the TRP-PLIK2dpolypeptide. For example, phosphorylation at specific sites may beinvolved in regulating the proteins ability to associate or bind toother molecules (e.g., proteins, ligands, substrates, DNA, etc.). In thepresent case, phosphorylation may modulate the ability of the TRP-PLIK2dpolypeptide to associate with other potassium channel alpha subunits,beta subunits, or its ability to modulate potassium channel function.

[0399] The TRP-PLIK2d polypeptide was predicted to comprise twenty ninePKC phosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. . . .260:12492-12499(1985); which are hereby incorporated by referenceherein.

[0400] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: IILSKSQKSWIKG(SEQ ID NO:214), STIIPSSKNPHRC (SEQ ID NO:215), SVEKHTTKSPTDT (SEQ IDNO:216), SHSSHSLRKIWTV (SEQ ID NO:217), LSVWETVKDKDPV (SEQ ID NO:218),VVCEGTGRAADLL (SEQ ID NO:219), DLLAFTHKHLADE (SEQ ID NO:220),NTFNFSLKQSKHL (SEQ ID NO:221), YRSNYTRKHFRAL (SEQ ID NO:222),IVLHKSRKKSKEQ (SEQ ID NO:223), HGEEATVKAVIAC (SEQ ID NO:224),DQNASSSKESASV (SEQ ID NO:225), SKESASVKEYDLE (SEQ ID NO:226),QHLPWTRKVYEFY (SEQ ID NO:227), EPGKFTQKVKVWI (SEQ ID NO:228),RKAILSPKEPPSW (SEQ ID NO:229), RIRVTSERVTEMY (SEQ ID NO:230),ALTVDTLKVLSAV (SEQ ID NO:231), KRKHSTCKKLPHS (SEQ ID NO:232),LEITNSKREATNV (SEQ ID NO:233), ETGVFSIKKKWQT (SEQ ID NO:234),TCDSDSSRSEQHQ (SEQ ID NO:235), SLSDNSTRSAQSS (SEQ ID NO:236),FARSHSFRFHKEE (SEQ ID NO:237), KDRRLSKKKKNTQ (SEQ ID NO:238),DKISASLKSPQEP (SEQ ID NO:239), SMSSWSQRGRAAM (SEQ ID NO:240),QTIPYTPRFLEVF (SEQ ID NO:241), and/or PPARETGRNSPED (SEQ ID NO:242).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these TRP-PLIK2d PKCphosphorylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0401] The present invention also encompasses immunogenic and/orantigenic epitopes of the TRP-PLIK2d polypeptide.

[0402] The TRP-PLIK2d polypeptide has been shown to comprise seventeenglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0403] Asparagine phosphorylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. . .. 265:11397-I1404(1990).

[0404] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:HGGIQNFTMPSKFK (SEQ ID NO:243), IQNTFNFSLKQSKH (SEQ ID NO:244),LLKGTNLSASEQLN (SEQ ID NO:245), RAYRSNYTRKHFRA (SEQ ID NO:246),SSGNRNESAESTLH (SEQ ID NO:247), KSKEQNVSDDPEST (SEQ ID NO:248),SEELKNYSKQFGQL (SEQ ID NO:249), TYELRNWSNSTCLK (SEQ ID NO:250),LRNWSNSTCLKLAV (SEQ ID NO:251), YYSDQNASSSKESA (SEQ ID NO:252),ISEYWNLTETVAIG (SEQ ID NO:253), KMEDVNCSCEERIR (SEQ ID NO:254),SSLSDNSTRSAQSS (SEQ ID NO:255), PWLQPNTSFWINPL (SEQ ID NO:256),ICKIKNLSGSSEIG (SEQ ID NO:257), QGVGENLTDPSVIK (SEQ ID NO:258), and/orSPERINSTFGLEIK (SEQ ID NO:259). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these TRP-PLIK2d asparagine glycosylation site polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

[0405] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:7 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5911 ofSEQ ID NO:7, b is an integer between 15 to 5925, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:7,and where b is greater than or equal to a+14. TABLE I ATCC 5' NT DepositTotal NT of Start Gene CDNA No. Z NT SEQ Seq of Codon 3' NT AA Seq TotalAA No. CloneID and Date Vector ID. No.X Clone of ORF of ORF ID No.Y ofORF 1. TRP- PTA- pCR4 1 6054 1 6051 2 2017 PLIK2 4175 Blunt- (LTRPC Mar.21, 2002 TOPO 6, gene 95; AL35479 5; BAC57) 2 TRP- N/A pCR4 3 5913 15910 4 1970 PLIK2b Blunt- (LTRPC TOPO 6, gene 95; AL35479 5; BAC57splice variant) 3. TRP- N/A pCR4 5 5820 1 5817 6 1939 PLIK2c Blunt-(LTRPC TOPO 6, gene 95; AL35479 5; BAC57 splice variant) 4. TRP- N/ApCR4 7 5925 1 5922 8 1974 PLIK2d Blunt- (LTRPC TOPO 6, gene 95; AL354795; BAC57 splice variant)

[0406] Table I summarizes the information corresponding to each “GeneNo.” described above. The nucleotide sequence identified as “NT SEQ IDNO:X” was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table 1 and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually several overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO:X.

[0407] The cDNA Clone ID was deposited on the date and given thecorresponding deposit number listed in “ATCC Deposit No:PTA-4175 andDate.” “Vector” refers to the type of vector contained in the cDNA CloneID. “Total NT Seq. Of Clone” refers to the total number of nucleotidesin the clone contig identified by “Gene No.” The deposited clone maycontain all or most of the sequence of SEQ ID NO:X. The nucleotideposition of SEQ ID NO:X of the putative start codon (methionine) isidentified as “5′ NT of Start Codon of ORF.” The translated amino acidsequence, beginning with the methionine, is identified as “AA SEQ IDNO:Y,” although other reading frames can also be easily translated usingknown molecular biology techniques. The polypeptides produced by thesealternative open reading frames are specifically contemplated by thepresent invention.

[0408] The total number of amino acids within the open reading frame ofSEQ ID NO:Yis identified as “Total AA of ORF”.

[0409] SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:1, 3, 5, 7, and/or 97 is useful for designingnucleic acid hybridization probes that will detect nucleic acidsequences contained in SEQ ID NO:1, 3, 5, 7, and/or 97 or the cDNAcontained in the deposited clone. These probes will also hybridize tonucleic acid molecules in biological samples, thereby enabling a varietyof forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:2, 4, 6, 8, and/or 98 may beused, for example, to generate antibodies which bind specifically toproteins containing the polypeptides and the proteins encoded by thecDNA clones identified in Table 1.

[0410] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidesmay cause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

[0411] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1, 3, 5, 7, and/or 97 and the predicted translated amino acidsequence identified as SEQ ID NO:2, 4, 6, 8, and/or 98, but also asample of plasmid DNA containing a cDNA of the invention deposited withthe ATCC, as set forth in Table 1. The nucleotide sequence of eachdeposited clone can readily be determined by sequencing the depositedclone in accordance with known methods. The predicted amino acidsequence can then be verified from such deposits. Moreover, the aminoacid sequence of the protein encoded by a particular clone can also bedirectly determined by peptide sequencing or by expressing the proteinin a suitable host cell containing the deposited cDNA, collecting theprotein, and determining its sequence.

[0412] The present invention also relates to the genes corresponding toSEQ ID NO:1, 3, 5, 7, and/or 97, SEQ ID NO:2, 4, 6, 8, and/or 98, or thedeposited clone. The corresponding gene can be isolated in accordancewith known methods using the sequence information disclosed herein. Suchmethods include preparing probes or primers from the disclosed sequenceand identifying or amplifying the corresponding gene from appropriatesources of genomic material.

[0413] Also provided in the present invention are species homologs,allelic variants, and/or orthologs. The skilled artisan could, usingprocedures well-known in the art, obtain the polynucleotide sequencecorresponding to full-length genes (including, but not limited to thefull-length coding region), allelic variants, splice variants,orthologs, and/or species homologues of genes corresponding to SEQ IDNO:1, 3, 5, 7, and/or 97, SEQ ID NO:2, 4, 6, 8, and/or 98, or adeposited clone, relying on the sequence from the sequences disclosedherein or the clones deposited with the ATCC. For example, allelicvariants and/or species homologues may be isolated and identified bymaking suitable probes or primers which correspond to the 5′, 3′, orinternal regions of the sequences provided herein and screening asuitable nucleic acid source for allelic variants and/or the desiredhomologue.

[0414] The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0415] The polypeptides may be in the form of the protein, or may be apart of a larger protein, such as a fusion protein (see below). It isoften advantageous to include an additional amino acid sequence whichcontains secretory or leader sequences, pro-sequences, sequences whichaid in purification, such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

[0416] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural, synthetic or recombinant sources using protocolsdescribed herein or otherwise known in the art, such as, for example,antibodies of the invention raised against the full-length form of theprotein.

[0417] The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:1, 3,5, 7, and/or 97, and/or a cDNA provided in ATCC Deposit No. Z:. Thepresent invention also provides a polypeptide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:2, 4,6, 8, and/or 98, and/or a polypeptide encoded by the cDNA provided inATCC Deposit No:PTA-4175. The present invention also providespolynucleotides encoding a polypeptide comprising, or alternativelyconsisting of the polypeptide sequence of SEQ ID NO:2, 4, 6, 8, and/or98, and/or a polypeptide sequence encoded by the cDNA contained in ATCCDeposit No:PTA-4175.

[0418] Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:1, 3, 5, 7, and/or 97, and/or a cDNA provided in ATCC DepositNo.: that is less than, or equal to, a polynucleotide sequence that is 5mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 megabasepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs inlength.

[0419] The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:1, 3, 5, 7, and/or 97, the sequence contained ina deposit, and/or the nucleic acid sequence encoding the sequencedisclosed as SEQ ID NO:2, 4, 6, 8, and/or 98.

[0420] The present invention also encompasses polynucleotides capable ofhybridizing, preferably under reduced stringency conditions, morepreferably under stringent conditions, and most preferably under highlystingent conditions, to polynucleotides described herein. Examples ofstringency conditions are shown in Table 2 below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringen cy conditions are at leastas stringent as, for example, conditions M-R. TABLE 2 HybridHyridization Wash Stringency Polynucleotide Length TemperatureTemperature Condition Hybrid ± (bp)‡ and Buffer† and Buffer† A DNA:DNA >or equal 65° C.; 1 × SSC − 65° C; to 50 or- 42° C.; 0.3 × SSC 1 × SSC,50% formamide B DNA:DNA <50 Tb*; 1 × SSC Tb*; 1 × SSC C DNA:RNA > orequal 67° C.; 1 × SSC − 67° C.; to 50 or- 45° C; 0.3 × SSC 1 × SSC, 50%formamide D DNA:RNA <50 Td*; 1 × SSC Td*; 1 × SSC E RNA:RNA > or equal70° C.; 1 × SSC − 70° C.; to 50 or- 50° C; 0.3 × SSC 1 × SSC, 50%formamide F RNA:RNA <50 Tf*; 1 × SSC Tf*; 1 × SSC G DNA:DNA > or equal65° C.; 4 × SSC − 65° C.; 1 × SSC to 50 or- 45° C.; 4 × SSC, 50%formamide H DNA:DNA <50 Th*; 4 × SSC Th*; 4 × SSC I DNA:RNA > or equal67° C.; 4 × SSC − 67° C.; 1 × SSC to 50 or- 45° C.; 4 × SSC, 50%formamide J DNA:RNA <50 Tj*; 4 × SSC Tj*; 4 × SSC K RNA:RNA > or equal70° C.; 4 × SSC − 67° C; 1 × SSC to 50 or- 40° C; 6 × SSC, 50% formamideL RNA:RNA <50 Tl*; 2 × SSC Tl*; 2 × SSC M DNA:DNA > or equal 50° C.; 4 ×SSC − 50° C; 2 × SSC to 50 or- 40° C. 6 × SSC, 50% formamide N DNA:DNA<50 Tn*; 6 × SSC Tn*; 6 × SSC O DNA:RNA > or equal 55° C.; 4 × SSC − 55°C.; 2 × SSC to 50 or- 42° C.; 6 × SSC, 50% formamide P DNA:RNA <50 Tp*;6 × SSC Tp*; 6 × SSC Q RNA:RNA > or equal 60° C.; 4 × SSC − 60° C.; 2 ×SSC to 50 or- 45° C.; 6 × SSC, 50% formamide R RNA:RNA <50 Tr*; 4 × SSCTr*; 4 × SSC #Methods of aligning two or more polynucleotide sequencesand/or determining the percent identity between two polynucleotidesequences are well known in the art (e.g., MegAlign program of theDNA*Star suite of programs, etc). #]) + 0.41 (% G + C) − (600/N), whereN is the number of bases in the hybrid, and [Na+]]is the concentrationof sodium ions in the hybridization buffer ([NA+] for 1 × SSC = .165 M).

[0421] ‡- The “hybrid length” is the anticipated length for thehybridized region(s) of the hybridizing polynucleotides. Whenhybridizing a polynucletotide of unknown sequence, the hybrid is assumedto be that of the hybridizing polynucleotide of the present invention.When polynucleotides of known sequence are hybridized, the hybrid lengthcan be determined by aligning the sequences of the polynucleotides andidentifying the region or regions of optimal sequence complementarity.Methods of aligning two or more polynucleotide sequences and/ordetermining the percent identity between two polynucleotide sequencesare well known in the art (e.g., MegAlign program of the DNA*Star suiteof programs, etc).

[0422]554 - SSPE (lxSSPE is 0.15M NaCl, lOmM NaH2PO4, and 1.25 mM EDTA,pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl anmd 15 mMsodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes after hybridization is complete. Thehydridizations and washes may additionally include 5×Denhardt's reagent,0.5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5%sodium pyrophosphate, and up to 50% formamide.

[0423] *Tb-Tr: The hybridization temperature for hybrids anticipated tobe less than 50 base pairs in length should be 5-10° C. less than themelting temperature Tm of the hybrids there Tm is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, Tm(° C.)=2(# of A +T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, Tm(°C.)=81.5+16.6(log₁₀[Na+])+0.41(%G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer ([NA+] for 1×SSC=0.165 M).

[0424] ±- The present invention encompasses the substitution of any one,or more DNA or RNA hybrid partners with either a PNA, or a modifiedpolynucleotide. Such modified polynucleotides are known in the art andare more particularly described elsewhere herein.

[0425] Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

[0426] Preferably, such hybridizing polynucleotides have at least 70%sequence identity (more preferably, at least 80% identity; and mostpreferably at least 90% or 95% identity) with the polynucleotide of thepresent invention to which they hybridize, where sequence identity isdetermined by comparing the sequences of the hybridizing polynucleotideswhen aligned so as to maximize overlap and identity while minimizingsequence gaps. The determination of identity is well known in the art,and discussed more specifically elsewhere herein.

[0427] The invention encompasses the application of PCR methodology tothe polynucleotide sequences of the present invention, the clonedeposited with the ATCC, and/or the cDNA encoding the polypeptides ofthe present invention. PCR techniques for the amplification of nucleicacids are described in U.S. Pat. No. 4,683,195 and Saiki et al.,Science, 239:487-491 (1988). PCR, for example, may include the followingsteps, of denaturation of template nucleic acid (if double-stranded),annealing of primer to target, and polymerization. The nucleic acidprobed or used as a template in the amplification reaction may begenomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specificsequences from genomic DNA, specific RNA sequence, and/or cDNAtranscribed from mRNA. References for the general use of PCR techniques,including specific method parameters, include Mullis et al., Cold SpringHarbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology,Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650,(1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds.,Innis et al., Academic Press, New York, (1990).

[0428] Polynucleotide and Polypeptide Variants

[0429] The present invention also encompases variants (e.g., allelicvariants, orthologs, etc.) of the polynucleotide sequence disclosedherein in SEQ ID NO:1, 3, 5, 7, and/or 97, the complementary strandthereto, and/or the cDNA sequence contained in the deposited clone.

[0430] The present invention also encompasses variants of thepolypeptide sequence, and/or fragments therein, disclosed in SEQ IDNO:2, 4, 6, 8, and/or 98, a polypeptide encoded by the polunucleotidesequence in SEQ ID NO:1, 3, 5, 7, and/or 97, and/or a polypeptideencoded by a cDNA in the deposited clone.

[0431] “Variant” refers to a polynucleotide or polypeptide differingfrom the polynucleotide or polypeptide of the present invention, butretaining essential properties thereof. Generally, variants are overallclosely similar, and, in many regions, identical to the polynucleotideor polypeptide of the present invention.

[0432] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising, or alternatively consisting of, apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding a TRP-PLIK2 relatedpolypeptide having an amino acid sequence as shown in the sequencelisting and described in SEQ ID NO:1, 3, 5, 7, and/or 97 or the cDNAcontained in ATCC Deposit No:PTA-4175; (b) a nucleotide sequenceencoding a mature TRP-PLIK2 related polypeptide having the amino acidsequence as shown in the sequence listing and described in SEQ ID NO:1,3, 5, 7, and/or 97 or the cDNA contained in ATCC Deposit No:PTA-4175;(c) a nucleotide sequence encoding a biologically active fragment of aTRP-PLIK2 related polypeptide having an amino acid sequence shown in thesequence listing and described in SEQ ID NO:1, 3, 5, 7, and/or 97 or thecDNA contained in ATCC Deposit No:PTA-4175; (d) a nucleotide sequenceencoding an antigenic fragment of a TRP-PLIK2 related polypeptide havingan amino acid sequence shown in the sequence listing and described inSEQ ID NO:1, 3, 5, 7, and/or 97 or the cDNA contained in ATCC DepositNo:PTA-4175; (e) a nucleotide sequence encoding a TRP-PLIK2 relatedpolypeptide comprising the complete amino acid sequence encoded by ahuman cDNA plasmid containined in SEQ ID NO:1, 3, 5, 7, and/or 97 or thecDNA contained in ATCC Deposit No:PTA-4175; (f) a nucleotide sequenceencoding a mature TRP-PLIK2 realted polypeptide having an amino acidsequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 3, 5,7, and/or 97 or the cDNA contained in ATCC Deposit No:PTA-4175; (g) anucleotide sequence encoding a biologically active fragement of aTRP-PLIK2 related polypeptide having an amino acid sequence encoded by ahuman cDNA plasmid contained in SEQ ID NO:1, 3, 5, 7, and/or 97 or thecDNA contained in ATCC Deposit No:PTA-4175; (h) a nucleotide sequenceencoding an antigenic fragment of a TRP-PLIK2 related polypeptide havingan amino acid sequence encoded by a human cDNA plasmid contained in SEQID NO:1, 3, 5, 7, and/or 97 or the cDNA contained in ATCC DepositNo:PTA-4175; (I) a nucleotide sequence complimentary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h),above.

[0433] The present invention is also directed to polynucleotidesequences which comprise, or alternatively consist of, a polynucleotidesequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,95.9%, 96%, 97%, 97.4%, 97.6%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any ofthe nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h),above. Polynucleotides encoded by these nucleic acid molecules are alsoencompassed by the invention. In another embodiment, the inventionencompasses nucleic acid molecule which comprise, or alternatively,consist of a polynucleotide which hybridizes under stringent conditions,or alternatively, under lower stringency conditions, to a polynucleotidein (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotideswhich hybridize to the complement of these nucleic acid molecules understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention, as arepolypeptides encoded by these polypeptides.

[0434] Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively, consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a TRP-PLIK2 related polypeptide having anamino acid sequence as shown in the sequence listing and described inTable 1; (b) a nucleotide sequence encoding a mature TRP-PLIK2 relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and described in Table 1; (c) a nucleotide sequence encoding abiologically active fragment of a TRP-PLIK2 related polypeptide havingan amino acid sequence as shown in the sequence listing and described inTable 1; (d) a nucleotide sequence encoding an antigenic fragment of aTRP-PLIK2 related polypeptide having an amino acid sequence as shown inthe sequence listing and described in Table 1; (e) a nucleotide sequenceencoding a TRP-PLIK2 related polypeptide comprising the complete aminoacid sequence encoded by a human cDNA in a cDNA plasmid contained in theATCC Deposit and described in Table 1; (f) a nucleotide sequenceencoding a mature TRP-PLIK2 related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table 1: (g) a nucleotide sequence encoding abiologically active fragment of a TRP-PLIK2 related polypeptide havingan amino acid sequence encoded by a human cDNA in a cDNA plasmidcontained in the ATCC Deposit and described in Table 1; (h) a nucleotidesequence encoding an antigenic fragment of a TRP-PLIK2 relatedpolypeptide having an amino acid sequence encoded by a human cDNA in acDNA plasmid contained in the ATCC deposit and described in Table 1; (i)a nucleotide sequence complimentary to any of the nucleotide sequencesin (a), (b), (c), (d), (e), (f), (g), or (h) above.

[0435] The present invention is also directed to nucleic acid moleculeswhich comprise, or alternatively, consist of, a nucleotide sequencewhich is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.9%,96%, 97%, 97.4%, 97.6%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h),above.

[0436] The present invention encompasses polypeptide sequences whichcomprise, or alternatively consist of, an amino acid sequence which isat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.8%, 96%, 97%,97.2%, 97.5%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to, the following non-limited examples, thepolypeptide sequence identified as SEQ ID NO:2, 4, 6, 8, and/or 98, thepolypeptide sequence encoded by a cDNA provided in the deposited clone,and/or polypeptide fragments of any of the polypeptides provided herein.Polynucleotides encoded by these nucleic acid molecules are alsoencompassed by the invention. In another embodiment, the inventionencompasses nucleic acid molecule which comprise, or alternatively,consist of a polynucleotide which hybridizes under stringent conditions,or alternatively, under lower stringency conditions, to a polynucleotidein (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotideswhich hybridize to the complement of these nucleic acid molecules understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention, as arepolypeptides encoded by these polypeptides.

[0437] The present invention is also directed to polypeptides whichcomprise, or alternatively consist of, an amino acid sequence which isat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.8%, 96%, 97%,97.2%, 97.5%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to, for example, the polypeptide sequenceshown in SEQ ID NO:2, 4, 6, 8, and/or 98, a polypeptide sequence encodedby the nucleotide sequence in SEQ ID NO:1, 3, 5, 7, and/or 97, apolypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/orpolypeptide fragments of any of these polypeptides (e.g., thosefragments described herein). Polynucleotides which hybridize to thecomplement of the nucleic acid molecules encoding these polypeptidesunder stringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompasses by the present invention, asare the polypeptides encoded by these polynucleotides.

[0438] By a nucleic acid having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thenucleic acid is identical to the reference sequence except that thenucleotide sequence may include up to five point mutations per each 100nucleotides of the reference nucleotide sequence encoding thepolypeptide. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence referenced in Table 1, the ORF (open reading frame),or any fragment specified as described herein.

[0439] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 95.8%, 95.9%, 96%, 97%, 97.2%, 97.4%, 97.5%, 97.6%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a nucleotide sequence of the present invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theCLUSTALW computer program (Thompson, J. D., et al., Nucleic AcidsResearch, 2(22):4673-4680, (1994)), which is based on the algorithm ofHiggins, D. G., et al., Computer Applications in the Biosciences(CABIOS), 8(2):189-191, (1992). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=l, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue SpecificGaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

[0440] The present invention encompasses the application of a manualcorrection to the percent identity results, in the instance where thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

[0441] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the CLUSTALW alignmentdoes not show a matched/alignment of the first 10 bases at 5′ end. The10 unpaired bases represent 10% of the sequence (number of bases at the5′ and 3′ ends not matched/total number of bases in the query sequence)so 10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

[0442] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino- or carboxy-terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0443] As a practical matter, whether any particular polypeptide is atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.8%, 96%, 97%,97.2%, 97.5%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to, for instance, an amino acid sequencereferenced in Table 1 (SEQ ID NO:2) or to the amino acid sequenceencoded by cDNA contained in a deposited clone, can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the CLUSTALW computerprogram (Thompson, J. D., et al., Nucleic Acids Research,2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D.G., et al., Computer Applications in the Biosciences (CABIOS),8(2):189-191, (1992). In a sequence alignment the query and subjectsequences are both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina CLUSTALW alignment of DNA sequences to calculate percent identity viapairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of TopDiagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap ExtensionPenalty=0.1, Scoring Method=Percent, Window Size=5 or the length of thesubject nucleotide sequence, whichever is shorter. For multiplealignments, the following CLUSTALW parameters are preferred: Gap OpeningPenalty=10; Gap Extension Parameter=0.05; Gap Separation PenaltyRange=8; End Gap Separation Penalty=Off; % Identity for AlignmentDelay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; andTransition Weighting=0. The pairwise and multple alignment parametersprovided for CLUSTALW above represent the default parameters as providedwith the AlignX software program (Vector NTI suite of programs, version6.0).

[0444] The present invention encompasses the application of a manualcorrection to the percent identity results, in the instance where thesubject sequence is shorter than the query sequence because of N- orC-terminal deletions, not because of internal deletions. If only thelocal pairwise percent identity is required, no manual correction isneeded. However, a manual correction may be applied to determine theglobal percent identity from a global polypeptide alignment. Percentidentity calculations based upon global polypeptide alignments are oftenpreferred since they reflect the percent identity between thepolypeptide molecules as a whole (i.e., including any polypeptideoverhangs, not just overlapping regions), as opposed to, only localmatching polypeptides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor N- and C-terminal truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the N-and C-termini, relative to the query sequence, the percent identity iscorrected by calculating the number of residues of the query sequencethat are N- and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what may be used for the purposes of the present invention.Only residues to the N- and C-termini of the subject sequence, which arenot matched/aligned with the query sequence, are considered for thepurposes of manually adjusting the percent identity score. That is, onlyquery residue positions outside the farthest N- and C-terminal residuesof the subject sequence.

[0445] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the CLUSTALW alignment does not show a matching/alignment of the first10 residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C- termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the CLUSTALWprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence, which are not matched/aligned withthe query. In this case the percent identity calculated by CLUSTALW isnot manually corrected. Once again, only residue positions outside theN- and C-terminal ends of the subject sequence, as displayed in theCLUSTALW alignment, which are not matched/aligned with the querysequence are manually corrected for. No other manual corrections arerequired for the purposes of the present invention.

[0446] In addition to the above method of aligning two or morepolynucleotide or polypeptide sequences to arrive at a percent identityvalue for the aligned sequences, it may be desirable in somecircumstances to use a modified version of the CLUSTALW algorithm whichtakes into account known structural features of the sequences to bealigned, such as for example, the SWISS-PROT designations for eachsequence. The result of such a modifed CLUSTALW algorithm may provide amore accurate value of the percent identity for two polynucleotide orpolypeptide sequences. Support for such a modified version of CLUSTALWis provided within the CLUSTALW algorithm and would be readilyappreciated to one of skill in the art of bioinformatics.

[0447] The variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in themRNA to those preferred by a bacterial host such as E. coli).

[0448] Naturally occurring variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level and are included inthe present invention. Alternatively, non-naturally occurring variantsmay be produced by mutagenesis techniques or by direct synthesis.

[0449] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

[0450] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem.268:22105-22111 (1993)) conducted extensive mutational analysis of humancytokine IL- a. They used random mutagenesis to generate over 3,500individual IL-la mutants that averaged 2.5 amino acid changes pervariant over the entire length of the molecule. Multiple mutations wereexamined at every possible amino acid position. The investigators foundthat “[m]ost of the molecule could be altered with little effect oneither [binding or biological activity].” In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0451] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N- or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

[0452] Alternatively, such N-terminus or C-terminus deletions of apolypeptide of the present invention may, in fact, result in asignificant increase in one or more of the biological activities of thepolypeptide(s). For example, biological activity of many polypeptidesare governed by the presence of regulatory domains at either one or bothtermini. Such regulatory domains effectively inhibit the biologicalactivity of such polypeptides in lieu of an activation event (e.g.,binding to a cognate ligand or receptor, phosphorylation, proteolyticprocessing, etc.). Thus, by eliminating the regulatory domain of apolypeptide, the polypeptide may effectively be rendered biologicallyactive in the absence of an activation event.

[0453] The invention further includes polypeptide variants that showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

[0454] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0455] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0456] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved.

[0457] The invention encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by the polypeptide of the presentinvention. Similarity is. determined by conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a polypeptide by another amino acid of like characteristics(e.g., chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0458] Tolerated conservative amino acid substitutions of the presentinvention involve replacement of the aliphatic or hydrophobic aminoacids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Serand Thr; replacement of the acidic residues Asp and Glu; replacement ofthe amide residues Asn and Gln, replacement of the basic residues Lys,Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp,and replacement of the small-sized amino acids Ala, Ser, Thr, Met, andGly.

[0459] In addition, the present invention also encompasses theconservative substitutions provided in Table III below. TABLE III ForAmino Acid Code Replace with any of: Alanine A D-Ala, Gly, beta-Ala,L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met,Ile, D- Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu,D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln,D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine QD-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, AcpIsoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile,D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa,His, D-His, Trp, D-Trp, Trans- 3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D-or L-1- oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

[0460] Aside from the uses described above, such amino acidsubstitutions may also increase protein or peptide stability. Theinvention encompasses amino acid substitutions that contain, forexample, one or more non-peptide bonds (which replace the peptide bonds)in the protein or peptide sequence. Also included are substitutions thatinclude amino acid residues other than naturally occurring L-aminoacids, e.g., D-amino acids or non-naturally occurring or synthetic aminoacids, e.g., β or γ amino acids.

[0461] Both identity and similarity can be readily calculated byreference to the following publications: Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Informatics Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress,New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0462] In addition, the present invention also encompasses substitutionof amino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

[0463] Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

[0464] Besides conservative amino acid substitution, variants of thepresent invention include, but are not limited to, the following: (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

[0465] For example, polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0466] Moreover, the invention further includes polypeptide variantscreated through the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:1, 3, 5, 7,and/or 97, the sequence of the clone submitted in a deposit, and/or thecDNA encoding the polypeptide disclosed as SEQ ID NO:2, 4, 6, 8, and/or98. Such DNA Shuffling technology is known in the art and moreparticularly described elsewhere herein (e.g., WPC, Stemmer, PNAS,91:10747, (1994)), and in the Examples provided herein).

[0467] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of the present invention havingan amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of the present invention, which contains atleast one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of thepresent invention or fragments thereof (e.g., the mature form and/orother fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or50-150, conservative amino acid substitutions are preferable.

[0468] Polynucleotide and Polypeptide Fragments

[0469] The present invention is directed to polynucleotide fragments ofthe polynucleotides of the invention, in addition to polypeptidesencoded therein by said polynucleotides and/or fragments.

[0470] In the present invention, a “polynucleotide fragment” refers to ashort polynucleotide having a nucleic acid sequence which: is a portionof that contained in a deposited clone, or encoding the polypeptideencoded by the cDNA in a deposited clone; is a portion of that shown inSEQ ID NO:1, 3, 5, 7, and/or 97 or the complementary strand thereto, oris a portion of a polynucleotide sequence encoding the polypeptide ofSEQ ID NO:2, 4, 6, 8, and/or 98. The nucleotide fragments of theinvention are preferably at least about 15 nt, and more preferably atleast about 20 nt, still more preferably at least about 30 nt, and evenmore preferably, at least about 40 nt, at least about 50 nt, at leastabout 75 nt, or at least about 150 nt in length. A fragment “at least 20nt in length,” for example, is intended to include 20 or more contiguousbases from the cDNA sequence contained in a deposited clone or thenucleotide sequence shown in SEQ ID NO:1, 3, 5, 7, and/or 97. In thiscontext “about” includes the particularly recited value, a value largeror smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus, or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

[0471] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-I100, 1101-I150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ IDNO:1, 3, 5, 7, and/or 97, or the complementary strand thereto, or thecDNA contained in a deposited clone. In this context “about” includesthe particularly recited ranges, and ranges larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.Preferably, these fragments encode a polypeptide which has biologicalactivity. More preferably, these polynucleotides can be used as probesor primers as discussed herein. Also encompassed by the presentinvention are polynucleotides which hybridize to these nucleic acidmolecules under stringent hybridization conditions or lower stringencyconditions, as are the polypeptides encoded by these polynucleotides.

[0472] In the present invention, a “polypeptide fragment” refers to anamino acid sequence which is a portion of that contained in SEQ ID NO:2,4, 6, 8, and/or 98 or encoded by the cDNA contained in a depositedclone. Protein (polypeptide) fragments may be “free-standing,” orcomprised within a larger polypeptide of which the fragment forms a partor region, most preferably as a single continuous region. Representativeexamples of polypeptide fragments of the invention, include, forexample, fragments comprising, or alternatively consisting of, fromabout amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120,121-140, 141-160, or 161 to the end of the coding region. Moreover,polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, or 150 amino acids in length. In this context“about” includes the particularly recited ranges or values, and rangesor values larger or smaller by several (5, 4, 3, 2, or 1) amino acids,at either extreme or at both extremes. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0473] Preferred polypeptide fragments include the full-length protein.Further preferred polypeptide fragments include the full-length proteinhaving a continuous series of deleted residues from the amino or thecarboxy terminus, or both. For example, any number of amino acids,ranging from 1-60, can be deleted from the amino terminus of thefull-length polypeptide. Similarly, any number of amino acids, rangingfrom 1-30, can be deleted from the carboxy terminus of the full-lengthprotein. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

[0474] Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:2, 4, 6, 8, and/or 98falling within conserved domains are specifically contemplated by thepresent invention. Moreover, polynucleotides encoding these domains arealso contemplated.

[0475] Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity. Polynucleotides encoding these polypeptidefragments are also encompassed by the invention.

[0476] In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

[0477] The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, 4, 6, 8, and/or 98, or an epitope ofthe polypeptide sequence encoded by a polynucleotide sequence containedin ATCC deposit No. Z or encoded by a polynucleotide that hybridizes tothe complement of the sequence of SEQ ID NO:1, 3, 5, 7, and/or 97 orcontained in ATCC deposit No. Z under stringent hybridization conditionsor lower stringency hybridization conditions as defined supra. Thepresent invention further encompasses polynucleotide sequences encodingan epitope of a polypeptide sequence of the invention (such as, forexample, the sequence disclosed in SEQ ID NO:1), polynucleotidesequences of the complementary strand of a polynucleotide sequenceencoding an epitope of the invention, and polynucleotide sequences whichhybridize to the complementary strand under stringent hybridizationconditions or lower stringency hybridization conditions defined supra.

[0478] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998- 4002 (1983)). The term “antigenicepitope,” as used herein, is defined as a portion of a protein to whichan antibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross- reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0479] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0480] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length, or longer. Additional non-exclusive preferredantigenic epitopes include the antigenic epitopes disclosed herein, aswell as portions thereof. Antigenic epitopes are useful, for example, toraise antibodies, including monoclonal antibodies, that specificallybind the epitope. Preferred antigenic epitopes include the antigenicepitopes disclosed herein, as well as any combination of two, three,four, five or more of these antigenic epitopes. Antigenic epitopes canbe used as the target molecules in immunoassays. (See, for instance,Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science219:660-666 (1983)).

[0481] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

[0482] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier- coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0483] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded ontoN12+nitriloacetic acid-agarose column and histidine-tagged proteins canbe selectively eluted with imidazole-containing buffers.

[0484] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each ofthese patents and publications are hereby incorporated by reference inits entirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:1, 3, 5, 7, and/or 97 and the polypeptidesencoded by these polynucleotides may be achieved by DNA shuffling. DNAshuffling involves the assembly of two or more DNA segments byhomologous or site-specific recombination to generate variation in thepolynucleotide sequence. In another embodiment, polynucleotides of theinvention, or the encoded polypeptides, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of a polynucleotide encoding a polypeptide of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

[0485] Antibodies

[0486] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ ID NO:2, 4, 6, 8,and/or 98, and/or an epitope, of the present invention (as determined byimmunoassays well known in the art for assaying specificantibody-antigen binding). Antibodies of the invention include, but arenot limited to, polyclonal, monoclonal, monovalent, bispecific,heteroconjugate, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) ismeant to include intact molecules, as well as, antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation ofthe animal or plant, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., J. Nucl. Med. . . . 24:316-325 (1983)).Thus, these fragments are preferred, as well as the products of a FAB orother immunoglobulin expression library. Moreover, antibodies of thepresent invention include chimeric, single chain, and humanizedantibodies.

[0487] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0488] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0489] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0490] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homologue of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In specific embodiments, antibodies of thepresent invention cross-react with murine, rat and/or rabbit homologuesof human proteins and the corresponding epitopes thereof. Antibodiesthat do not bind polypeptides with less than 95%, less than 90%, lessthan 85%, less than 80%, less than 75%, less than 70%, less than 65%,less than 60%, less than 55%, and less than 50% identity (as calculatedusing methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention. In aspecific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic polypeptide, orcombination(s) of 2, 3, 4, 5, or more of the specific antigenic and/orimmunogenic polypeptides disclosed herein. Further included in thepresent invention are antibodies which bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6M, 5×10-7 M, 107 M,5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-I1M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15M, or 10-15 M.

[0491] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0492] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0493] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-I1301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-I167 (1998); Bartunek et al., Cytokine 8(l):14-20 (1996)(which are all incorporated by reference herein in their entireties).

[0494] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0495] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

[0496] The antibodies of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent 10 attachment does not prevent theantibody from generating an anti-idiotypic response. For example, butnot by way of limitation, the antibody derivatives include antibodiesthat have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited tospecific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

[0497] The antibodies of the present invention may be generated by anysuitable method known in the art.

[0498] The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988); and CurrentProtocols, Chapter 2; which are hereby incorporated herein by referencein its entirety). In a preferred method, a preparation of the TRP-PLIK2polypeptides, TRP-PLIK2b, TRP-PLIK2c, and/or TRP-PLIK2d protein isprepared and purified to render it substantially free of naturalcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. The administration of the polypeptides of the presentinvention may entail one or more injections of an immunizing agent and,if desired, an adjuvant. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art. For the purposesof the invention, “immunizing agent” may be defined as a polypeptide ofthe invention, including fragments, variants, and/or derivativesthereof, in addition to fusions with heterologous polypeptides and otherforms of the polypeptides described herein.

[0499] Typically, the immunizing agent and/or adjuvant will be injectedin the mammal by multiple subcutaneous or intraperitoneal injections,though they may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0500] The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., pp. 563-681 (1981); Kohler et al., Eur. J.Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976), orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies includes, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

[0501] In a hybridoma method, a mouse, a humanized mouse, a mouse with ahuman immune system, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

[0502] The immunizing agent will typically include polypeptides of thepresent invention or a fusion protein thereof. Preferably, theimmunizing agent consists of an TRP-PLIK2 polypeptides, TRP-PLIK2b,TRP-PLIK2c, and/or TRP-PLIK2d polypeptide or, more preferably, with aTRP-PLIK2 polypeptides, TRP-PLIK2b, TRP-PLIK2c, and/or TRP-PLIK2dpolypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 degrees C.), and supplemented with about 10 g/lof nonessential amino acids, about 1,000 U/ml of penicillin, and about100 ug/ml of streptomycin. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986), pp. 59-103). Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0503] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, California and the American Type CultureCollection, Manassas, Virginia. More preferred are the parent myelomacell line (SP2O) as provided by the ATCC. As inferred throughout thespecification, human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

[0504] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst the polypeptides of the present invention. Preferably, thebinding specificity of monoclonal antibodies produced by the hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbantassay (ELISA). Such techniques are known in the art and within the skillof the artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

[0505] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods (Goding, supra, and/or according to Wands et al.(Gastroenterology 80:225-232 (1981)). Suitable culture media for thispurpose include, for example, Dulbecco's Modified Eagle's Medium andRPM1-1640. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0506] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-sepharose, hydroxyapatite chromatography, gelexclusion chromatography, gel electrophoresis, dialysis, or affinitychromatography.

[0507] The skilled artisan would acknowledge that a variety of methodsexist in the art for the production of monoclonal antibodies and thus,the invention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hydridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal, supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

[0508] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0509] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art. Monoclonal antibodies can be prepared usinga wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981) (said references incorporated by reference in their entireties).The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

[0510] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples described herein. In a non-limitingexample, mice can be immunized with a polypeptide of the invention or acell expressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

[0511] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0512] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0513] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0514] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

[0515] For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabillyet al., Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985);U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which areincorporated herein by reference in their entirety. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and a framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

[0516] In general, the humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992).

[0517] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety. The techniques of coleet al., and Boerder et al., are also available for the preparation ofhuman monoclonal antibodies (cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

[0518] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Pat. No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

[0519] Similarly, human antibodies can be made by introducing humanimmunoglobulin, loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., Biotechnol., 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al.,Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol.,14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93(1995).

[0520] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0521] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0522] Such anti-idiotypic antibodies capable of binding to theTRP-PLIK2 polypeptides, TRP-PLIK2b, TRP-PLIK2c, and/or TRP-PLIK2dpolypeptide can be produced in a two-step procedure. Such a method makesuse of the fact that antibodies are themselves antigens, and therefore,it is possible to obtain an antibody that binds to a second antibody. Inaccordance with this method, protein specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones that produce an antibody whose abilityto bind to the protein-specific antibody can be blocked by thepolypeptide. Such antibodies comprise anti-idiotypic antibodies to theprotein-specific antibody and can be used to immunize an animal toinduce formation of further protein-specific antibodies.

[0523] The antibodies of the present invention may be bispecificantibodies. Bispecific antibodies are monoclonal, Preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present invention, one of the bindingspecificities may be directed towards a polypeptide of the presentinvention, the other may be for any other antigen, and preferably for acell-surface protein, receptor, receptor subunit, tissue-specificantigen, virally derived protein, virally encoded envelope protein,bacterially derived protein, or bacterial surface protein, etc.

[0524] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published May13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0525] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CHI) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

[0526] Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for the treatment of HIV infection (WO 91/00360; WO 92/20373; andEP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0527] Polynucleotides Encoding Antibodies

[0528] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2, 4, 6, 8, and/or 98.

[0529] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0530] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0531] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties ), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0532] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0533] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0534] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423- 42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038- 1041 (1988)).

[0535] More preferably, a clone encoding an antibody of the presentinvention may be obtained according to the method described in theExample section herein.

[0536] Methods of Producing Antibodies

[0537] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0538] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0539] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0540] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0541] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0542] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0543] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non- essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. NatI. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0544] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0545] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0546] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0547] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0548] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0549] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0550] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0551] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337- 11341(1992) (said references incorporated by reference intheir entireties).

[0552] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:2, 4, 6, 8,and/or 98 may be fused or conjugated to the above antibody portions toincrease the in vivo half life of the polypeptides or for use inimmunoassays using methods known in the art. Further, the polypeptidescorresponding to SEQ ID NO:2, 4, 6, 8, and/or 98 may be fused orconjugated to the above antibody portions to facilitate purification.One reported example describes chimeric proteins consisting of the firsttwo domains of the human CD4-polypeptide and various domains of theconstant regions of the heavy or light chains of mammalianimmunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86(1988). The polypeptides of the present invention fused or conjugated toan antibody having disulfide- linked dimeric structures (due to the IgG)may also be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases,the Fc part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hlL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hlL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

[0553] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

[0554] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 1251, 1311, 111In or 99Tc.

[0555] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

[0556] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, 13-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0557] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0558] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0559] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0560] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0561] The present invention also encompasses the creation of syntheticantibodies directed against the polypeptides of the present invention.One example of synthetic antibodies is described in Radrizzani, M., etal., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class ofsynthetic antibodies has been described and are referred to asmolecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies,peptides, and enzymes are often used as molecular recognition elementsin chemical and biological sensors. However, their lack of stability andsignal transduction mechanisms limits their use as sensing devices.Molecularly imprinted polymers (MIPs) are capable of mimicking thefunction of biological receptors but with less stability constraints.Such polymers provide high sensitivity and selectivity while maintainingexcellent thermal and mechanical stability. MIPs have the ability tobind to small molecules and to target molecules such as organics andproteins′ with equal or greater potency than that of natural antibodies.These “super” MIPs have higher affinities for their target and thusrequire lower concentrations for efficacious binding.

[0562] During synthesis, the MIPs are imprinted so as to havecomplementary size, shape, charge and functional groups of the selectedtarget by using the target molecule itself (such as a polypeptide,antibody, etc.), or a substance having a very similar structure, as its“print” or “template.” MIPs can be derivatized with the same reagentsafforded to antibodies. For example, fluorescent 'super′ MIPs can becoated onto beads or wells for use in highly sensitive separations orassays, or for use in high throughput screening of proteins.

[0563] Moreover, MIPs based upon the structure of the polypeptide(s) ofthe present invention may be useful in screening for compounds that bindto the polypeptide(s) of the invention. Such a MIP would serve the roleof a synthetic “receptor” by minimicking the native architecture of thepolypeptide. In fact, the ability of a MIP to serve the role of asynthetic receptor has already been demonstrated for the estrogenreceptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001);Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71,(2001)). A synthetic receptor may either be mimicked in its entirety(e.g., as the entire protein), or mimicked as a series of short peptidescorresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys,Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may beemployed in any one or more of the screening methods described elsewhereherein.

[0564] MIPs have also been shown to be useful in “sensing” the presenceof its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens,Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J.L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J.L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed usinga polypeptide of the present invention may be used in assays designed toidentify, and potentially quantitate, the level of said polypeptide in asample. Such a MIP may be used as a substitute for any componentdescribed in the assays, or kits, provided herein (e.g., ELISA, etc.).

[0565] A number of methods may be employed to create MIPs to a specificreceptor, ligand, polypeptide, peptide, organic molecule. Severalpreferred methods are described by Esteban et al in J. Anal, Chem.,370(7):795-802, (2001), which-is hereby incorporated herein by referencein its entirety in addition to any references cited therein. Additionalmethods are known in the art and are encompassed by the presentinvention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem,Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J.,De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54,(2001); which are hereby incorporated by reference in their entiretyherein.

[0566] Uses for Antibodies Directed Against Polypeptides of theInvention

[0567] The antibodies of the present invention have various utilities.For example, such antibodies may be used in diagnostic assays to detectthe presence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

[0568] Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), ppl47-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 1251, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

[0569] Antibodies directed against the polypeptides of the presentinvention are useful for the affinity purification of such polypeptidesfrom recombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

[0570] Immunophenotyping

[0571] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0572] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

[0573] Assays For Antibody Binding

[0574] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0575] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0576] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%- 20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 1251) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0577] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0578] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 1251) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., 3H or 1251) in the presence of increasingamounts of an unlabeled second antibody.

[0579] Therapeutic Uses Of Antibodies

[0580] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0581] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0582] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0583] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0584] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-2 M,10-2 M, 5×10-3 M, 10-3 M, 5 X 10-4 M, 10-4 M, 5×10-5 M, 10-5M, 5×10-6 M,10-6 M, 5×10-7 M, 10-7 M, 5 X 10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10M, 10-10 M, 5×10-I1 M, 10-I1 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10- 13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

[0585] Antibodies directed against polypeptides of the present inventionare useful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

[0586] Likewise, one could envision cloning the gene encoding anantibody directed against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

[0587] Alternatively, antibodies of the present invention could beproduced in a plant (e.g., cloning the gene of the antibody directedagainst a polypeptide of the present invention, and transforming a plantwith a suitable vector comprising said gene for constitutive expressionof the antibody within the plant), and the plant subsequently ingestedby an animal, thereby conferring temporary immunity to the animal forthe specific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

[0588] In another embodiment, antibodies of the present invention,preferably polyclonal antibodies, more preferably monoclonal antibodies,and most preferably single-chain antibodies, can be used as a means ofinhibiting gene expression of a particular gene, or genes, in a human,mammal, and/or other organism. See, for example, InternationalPublication Number WO 00/05391, published 2/3/00, to Dow AgrosciencesLLC. The application of such methods for the antibodies of the presentinvention are known in the art, and are more particularly describedelsewhere herein.

[0589] In yet another embodiment, antibodies of the present inventionmay be useful for multimerizing the polypeptides of the presentinvention. For example, certain proteins may confer enhanced biologicalactivity when present in a multimeric state (i.e., such enhancedactivity may be due to the increased effective concentration of suchproteins whereby more protein is available in a localized location).

[0590] Antibody-based Gene Therapy

[0591] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0592] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0593] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0594] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue- specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0595] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid- carrying vectors, or indirect, in which case,cells are first transformed with the nucleic acids in vitro, thentransplanted into the patient. These two approaches are known,respectively, as in vivo or ex vivo gene therapy.

[0596] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0597] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdrl gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0598] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143- 155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0599] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0600] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0601] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0602] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0603] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0604] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0605] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0606] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

[0607] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0608] Therapeutic/Prophylactic Administration and Compositions

[0609] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0610] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0611] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0612] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0613] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0614] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Florida (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0615] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0616] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0617] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0618] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0619] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0620] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0621] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0622] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0623] Diagnosis and Imaging With Antibodies

[0624] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

[0625] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0626] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0627] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0628] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0629] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0630] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0631] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0632] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0633] Kits

[0634] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0635] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0636] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0637] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0638] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

[0639] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0640] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0641] Fusion Proteins

[0642] Any polypeptide of the present invention can be used to generatefusion proteins. For example, the polypeptide of the present invention,when fused to a second protein, can be used as an antigenic tag.Antibodies raised against the polypeptide of the present invention canbe used to indirectly detect the second protein by binding to thepolypeptide. Moreover, because certain proteins target cellularlocations based on trafficking signals, the polypeptides of the presentinvention can be used as targeting molecules once fused to otherproteins.

[0643] Examples of domains that can be fused to polypeptides of thepresent invention include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

[0644] Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. Similarly, peptidecleavage sites can be introduced in-between such peptide moieties, whichcould additionally be subjected to protease activity to remove saidpeptide(s) from the protein of the present invention. The addition ofpeptide moieties, including peptide cleavage sites, to facilitatehandling of polypeptides are familiar and routine techniques in the art.

[0645] Moreover, polypeptides of the present invention, includingfragments, and specifically epitopes, can be combined with parts of theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3, and any combination thereof, including bothentire domains and portions thereof), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995).)

[0646] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of the constantregion of immunoglobulin molecules together with another human proteinor part thereof. In many cases, the Fc part in a fusion protein isbeneficial in therapy and diagnosis, and thus can result in, forexample, improved pharmacokinetic properties. (EP-A 0232 262.)Alternatively, deleting the Fc part after the fusion protein has beenexpressed, detected, and purified, would be desired. For example, the Fcportion may hinder therapy and diagnosis if the fusion protein is usedas an antigen for immunizations. In drug discovery, for example, humanproteins, such as hIL-5, have been fused with Fc portions for thepurpose of high-throughput screening assays to identify antagonists ofhIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995);K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

[0647] Moreover, the polypeptides of the present invention can be fusedto marker sequences (also referred to as “tags”). Due to theavailability of antibodies specific to such “tags”, purification of thefused polypeptide of the invention, and/or its identification issignificantly facilitated since antibodies specific to the polypeptidesof the invention are not required. Such purification may be in the formof an affinity purification whereby an anti-tag antibody or another typeof affinity matrix (e.g., anti-tag antibody attached to the matrix of aflow-thru column) that binds to the epitope tag is present. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984)).

[0648] The skilled artisan would acknowledge the existence of other“tags” which could be readily substituted for the tags referred to suprafor purification and/or identification of polypeptides of the presentinvention (Jones C., et al., J Chromatogr A. 707(l):3-22 (1995)). Forexample, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990), the Flag-peptide - i.e., the octapeptide sequence DYKDDDDK (SEQID NO:261), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitopepeptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); theT7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci.USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFPepitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

[0649] The present invention also encompasses the attachment of up tonine codons encoding a repeating series of up to nine arginine aminoacids to the coding region of a polynucleotide of the present invention.The invention also encompasses chemically derivitizing a polypeptide ofthe present invention with a repeating series of up to nine arginineamino acids. Such a tag, when attached to a polypeptide, has recentlybeen shown to serve as a universal pass, allowing compounds access tothe interior of cells without additional derivitization or manipulation(Wender, P., et al., unpublished data).

[0650] Protein fusions involving polypeptides of the present invention,including fragments and/or variants thereof, can be used for thefollowing, non-limiting examples, subcellular localization of proteins,determination of protein-protein interactions via immunoprecipitation,purification of proteins via affinity chromatography, functional and/orstructural characterization of protein. The present invention alsoencompasses the application of hapten specific antibodies for any of theuses referenced above for epitope fusion proteins. For example, thepolypeptides of the present invention could be chemically derivatized toattach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to theavailability of monoclonal antibodies specific to such haptens, theprotein could be readily purified using immunoprecipation, for example.

[0651] Polypeptides of the present invention, including fragments and/orvariants thereof, in addition to, antibodies directed against suchpolypeptides, fragments, and/or variants, may be fused to any of anumber of known, and yet to be deterrnined, toxins, such as ricin,saporin (Mashiba H, et al., Ann. N. Y. Acad. Sci. 1999;886:233-5), or HCtoxin (Tonukari NJ, et al., Plant Cell. 2000 Feb;12(2):237-248), forexample. Such fusions could be used to deliver the toxins to desiredtissues for which a ligand or a protein capable of binding to thepolypeptides of the invention exists.

[0652] The invention encompasses the fusion of antibodies directedagainst polypeptides of the present invention, including variants andfragments thereof, to said toxins for delivering the toxin to specificlocations in a cell, to specific tissues, and/or to specific species.Such bifunctional antibodies are known in the art, though a reviewdescribing additional advantageous fusions, including citations formethods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm.11:548-557, (1999); this publication, in addition to the referencescited therein, are hereby incorporated by reference in their entiretyherein. In this context, the term “toxin” may be expanded to include anyheterologous protein, a small molecule, radionucleotides, cytotoxicdrugs, liposomes, adhesion molecules, glycoproteins, ligands, cell ortissue-specific ligands, enzymes, of bioactive agents, biologicalresponse modifiers, anti-fungal agents, hormones, steroids, vitamins,peptides, peptide analogs, anti-allergenic agents, anti-tubercularagents, anti-viral agents, antibiotics, anti-protozoan agents, chelates,radioactive particles, radioactive ions, X-ray contrast agents,monoclonal antibodies, polyclonal antibodies and genetic material. Inview of the present disclosure, one skilled in the art could determinewhether any particular “toxin” could be used in the compounds of thepresent invention. Examples of suitable “toxins” listed above areexemplary only and are not intended to limit the “toxins” that may beused in the present invention.

[0653] Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

[0654] Vectors, Host Cells, and Protein Production

[0655] The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0656] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0657] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0658] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

[0659] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYDI, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S I, pPIC3.5K, pPIC9K, andPA0815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

[0660] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

[0661] A polypeptide of this invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0662] Polypeptides of the present invention, and preferably thesecreted form, can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

[0663] In one embodiment, the yeast Pichia pastoris is used to expressthe polypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using 02. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for 02. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

[0664] In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D.R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOXI promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

[0665] Many other yeast vectors could be used in place of pPIC9K, suchas, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG, as required.

[0666] In another embodiment, high-level expression of a heterologouscoding sequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, andgrowing the yeast culture in the absence of methanol.

[0667] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with the polynucleotides of the invention,and which activates, alters, and/or amplifies endogenouspolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous polynucleotide sequences via homologousrecombination, resulting in the formation of a new transcription unit(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.5,733,761, issued Mar. 31, 1998; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

[0668] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y., and Hunkapiller et al., Nature, 10 310:105-I11 (1984)). Forexample, a polypeptide corresponding to a fragment of a polypeptidesequence of the invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the polypeptide sequence. Non-classical amino acidsinclude, but are not limited to, to the D-isomers of the common aminoacids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyricacid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid,Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine,norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0669] The invention encompasses polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

[0670] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or 0-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or 0-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein, the addition ofepitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin,maltose binding protein, etc.), attachment of affinity tags such asbiotin and/or streptavidin, the covalent attachment of chemical moietiesto the amino acid backbone, N- or C-terminal processing of thepolypeptides ends (e.g., proteolytic processing), deletion of theN-terminal methionine residue, etc.

[0671] Also provided by the invention are chemically modifiedderivatives of the polypeptides of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

[0672] The invention further encompasses chemical derivitization of thepolypeptides of the present invention, preferably where the chemical isa hydrophilic polymer residue. Exemplary hydrophilic polymers, includingderivatives, may be those that include polymers in which the repeatingunits contain one or more hydroxy groups (polyhydroxy polymers),including, for example, poly(vinyl alcohol); polymers in which therepeating units contain one or more amino groups (polyamine polymers),including, for example, peptides, polypeptides, proteins andlipoproteins, such as albumin and natural lipoproteins; polymers inwhich the repeating units contain one or more carboxy groups(polycarboxy polymers), including, for example, carboxymethylcellulose,alginic acid and salts thereof, such as sodium and calcium alginate,glycosaminoglycans and salts thereof, including salts of hyaluronicacid, phosphorylated and sulfonated derivatives of carbohydrates,genetic material, such as interleukin-2 and interferon, andphosphorothioate oligomers; and polymers in which the repeating unitscontain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates.

[0673] The molecular weight of the hydrophilic polymers may vary, and isgenerally about 50 to about 5,000,000, with polymers having a molecularweight of about 100 to about 50,000 being preferred. The polymers may bebranched or unbranched. More preferred polymers have a molecular weightof about 150 to about 10,000, with molecular weights of 200 to about8,000 being even more preferred.

[0674] For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0675] Additional preferred polymers which may be used to derivatizepolypeptides of the invention, include, for example, poly(ethyleneglycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate andpoly(vinyl alcohol), with PEG polymers being particularly preferred.Preferred among the PEG polymers are PEG polymers having a molecularweight of from about 100 to about 10,000. More preferably, the PEGpolymers have a molecular weight of from about 200 to about 8,000, withPEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of2,000, 5,000 and 8,000, respectively, being even more preferred. Othersuitable hydrophilic polymers, in addition to those exemplified above,will be readily apparent to one skilled in the art based on the presentdisclosure. Generally, the polymers used may include polymers that canbe attached to the polypeptides of the invention via alkylation oracylation reactions.

[0676] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0401384, herein incorporated by reference (coupling PEG to G-CSF), seealso Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reportingpegylation of GM-CSF using tresyl chloride). For example, polyethyleneglycol may be covalently bound through amino acid residues via areactive group, such as, a free amino or carboxyl group. Reactive groupsare those to which an activated polyethylene glycol molecule may bebound. The amino acid residues having a free amino group may includelysine residues and the N-terminal amino acid residues; those having afree carboxyl group may include aspartic acid residues glutamic acidresidues and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecules. Preferred for therapeutic purposes is attachment at an aminogroup, such as attachment at the N-terminus or lysine group.

[0677] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminus) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0678] As with the various polymers exemplified above, it iscontemplated that the polymeric residues may contain functional groupsin addition, for example, to those typically involved in linking thepolymeric residues to the polypeptides of the present invention. Suchfunctionalities include, for example, carboxyl, amine, hydroxy and thiolgroups. These functional groups on the polymeric residues can be furtherreacted, if desired, with materials that are generally reactive withsuch functional groups and which can assist in targeting specifictissues in the body including, for example, diseased tissue. Exemplarymaterials which can be reacted with the additional functional groupsinclude, for example, proteins, including antibodies, carbohydrates,peptides, glycopeptides, glycolipids, lectins, and nucleosides.

[0679] In addition to residues of hydrophilic polymers, the chemicalused to derivatize the polypeptides of the present invention can be asaccharide residue. Exemplary saccharides which can be derived include,for example, monosaccharides or sugar alcohols, such as erythrose,threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitoland sedoheptulose, with preferred monosaccharides being fructose,mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides,such as lactose, sucrose, maltose and cellobiose. Other saccharidesinclude, for example, inositol and ganglioside head groups. Othersuitable saccharides, in addition to those exemplified above, will bereadily apparent to one skilled in the art based on the presentdisclosure. Generally, saccharides which may be used for derivitizationinclude saccharides that can be attached to the polypeptides of theinvention via alkylation or acylation reactions.

[0680] Moreover, the invention also encompasses derivitization of thepolypeptides of the present invention, for example, with lipids(including cationic, anionic, polymerized, charged, synthetic,saturated, unsaturated, and any combination of the above, etc.).stabilizing agents.

[0681] The invention encompasses derivitization of the polypeptides ofthe present invention, for example, with compounds that may serve astabilizing function (e.g., to increase the polypeptides half-life insolution, to make the polypeptides more water soluble, to increase thepolypeptides hydrophilic or hydrophobic character, etc.). Polymersuseful as stabilizing materials may be of natural, semi-synthetic(modified natural) or synthetic origin. Exemplary natural polymersinclude naturally occurring polysaccharides, such as, for example,arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans,xylans (such as, for example, inulin), levan, fucoidan, carrageenan,galatocarolose, pectic acid, pectins, including amylose, pullulan,glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose,polyglucose, polydextrose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum,starch and various other natural homopolymer or heteropolymers, such asthose containing one or more of the following aldoses, ketoses, acids oramines: erythose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronicacid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof Accordingly, suitable polymers include, for example,proteins, such as albumin, polyalginates, and polylactide-coglycolidepolymers. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites,fluoroapatite polymers, polyethylenes (such as, for example,polyethylene glycol (including for example, the class of compoundsreferred to as Pluronics.RTM., commercially available from BASF,Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate),polypropylenes (such as, for example, polypropylene glycol),polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon,polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof. Methods for thepreparation of derivatized polypeptides of the invention which employpolymers as stabilizing compounds will be readily apparent to oneskilled in the art, in view of the present disclosure, when coupled withinformation known in the art, such as that described and referred to inUnger, U.S. Pat. No. 5,205,290, the disclosure of which is herebyincorporated by reference herein in its entirety.

[0682] Moreover, the invention encompasses additional modifications ofthe polypeptides of the present invention. Such additional modificationsare known in the art, and are specifically provided, in addition tomethods of derivitization, etc., in U.S. Pat. No. 6,028,066, which ishereby incorporated in its entirety herein.

[0683] The polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of thepolypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

[0684] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only polypeptides corresponding to the amino acid sequence ofSEQ ID NO:2, 4, 6, 8, and/or 98 or encoded by the cDNA contained in adeposited clone (including fragments, variants, splice variants, andfusion proteins, corresponding to these polypeptides as describedherein). These homomers may contain polypeptides having identical ordifferent amino acid sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only polypeptides having anidentical amino acid sequence. In another specific embodiment, a homomerof the invention is a multimer containing polypeptides having differentamino acid sequences. In specific embodiments, the multimer of theinvention is a homodimer (e.g., containing polypeptides having identicalor different amino acid sequences) or a homotrimer (e.g., containingpolypeptides having identical and/or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

[0685] As used herein, the term heteromer refers to a multimercontaining one or more heterologous polypeptides (i.e., polypeptides ofdifferent proteins) in addition to the polypeptides of the invention. Ina specific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

[0686] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

[0687] In one example, covalent associations are between theheterologous sequence contained in a fusion protein of the invention(see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained inan Fc fusion protein of the invention (as described herein). In anotherspecific example, covalent associations of fusion proteins of theinvention are between heterologous polypeptide sequence from anotherprotein that is capable of forming covalently associated multimers, suchas for example, osteoprotegerin (see, e.g., International PublicationNO: WO 98/49305, the contents of which are herein incorporated byreference in its entirety). In another embodiment, two or morepolypeptides of the invention are joined through peptide linkers.Examples include those peptide linkers described in U.S. Pat. No.5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple polypeptides of the invention separated by peptide linkers maybe produced using conventional recombinant DNA technology.

[0688] Another method for preparing multimer polypeptides of theinvention involves use of polypeptides of the invention fused to aleucine zipper or isoleucine zipper polypeptide sequence. Leucine zipperand isoleucine zipper domains are polypeptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, (1988)), and have since been found in avariety of different proteins. Among the known leucine zippers arenaturally occurring peptides and derivatives thereof that dimerize ortrimerize. Examples of leucine zipper domains suitable for producingsoluble multimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art.

[0689] Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

[0690] In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

[0691] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

[0692] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,polypeptides contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain (orhydrophobic or signal peptide) and which can be incorporated by membranereconstitution techniques into liposomes (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).

[0693] In addition, the polynucleotide insert of the present invention,could be operatively linked to “artificial” or chimeric promoters andtranscription factors. Specifically, the artificial promoter couldcomprise, or alternatively consist, of any combination of cis-acting DNAsequence elements that are recognized by trans-acting transcriptionfactors. Preferably, the cis acting DNA sequence elements andtrans-acting transcription factors are operable in mammals. Further, thetrans-acting transcription factors of such “artificial” promoters couldalso be “artificial” or chimeric in design themselves and could act asactivators or repressors to said “artificial” promoter.

[0694] Uses of the Polynucleotides

[0695] Each of the polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0696] The polynucleotides of the present invention are useful forchromosome identification. There exists an ongoing need to identify newchromosome markers, since few chromosome marking reagents, based onactual sequence data (repeat polymorphisms), are presently available.Each polynucleotide of the present invention can be used as a chromosomemarker.

[0697] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1,3, 5, 7, and/or 97. Primers can be selected using computer analysis sothat primers do not span more than one predicted exon in the genomicDNA. These primers are then used for PCR screening of somatic cellhybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the SEQ ID NO:1, 3, 5, 7,and/or 97 will yield an amplified fragment.

[0698] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

[0699] Precise chromosomal location of the polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

[0700] For chromosome mapping, the polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

[0701] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. Diseasemapping data are known in the art. Assuming 1 megabase mappingresolution and one gene per 20 kb, a cDNA precisely localized to achromosomal region associated with the disease could be one of 50-500potential causative genes.

[0702] Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected organisms can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected organisms, but not in normal organisms,indicates that the mutation may cause the disease. However, completesequencing of the polypeptide and the corresponding gene from severalnormal organisms is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

[0703] Furthermore, increased or decreased expression of the gene inaffected organisms as compared to unaffected organisms can be assessedusing polynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

[0704] Thus, the invention also provides a diagnostic method usefulduring diagnosis of a disorder, involving measuring the expression levelof polynucleotides of the present invention in cells or body fluid froman organism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

[0705] By “measuring the expression level of a polynucleotide of thepresent invention” is intended qualitatively or quantitatively measuringor estimating the level of the polypeptide of the present invention orthe level of the mRNA encoding the polypeptide in a first biologicalsample either directly (e.g., by determining or estimating absoluteprotein level or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

[0706] By “biological sample” is intended any biological sample obtainedfrom an organism, body fluids, cell line, tissue culture, or othersource which contains the polypeptide of the present invention or mRNA.As indicated, biological samples include body fluids (such as thefollowing non-limiting examples, sputum, amniotic fluid, urine, saliva,breast milk, secretions, interstitial fluid, blood, serum, spinal fluid,etc.) which contain the polypeptide of the present invention, and othertissue sources found to express the polypeptide of the presentinvention. Methods for obtaining tissue biopsies and body fluids fromorganisms are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

[0707] The method(s) provided above may Preferably be applied in adiagnostic method and/or kits in which polynucleotides and/orpolypeptides are attached to a solid support. In one exemplary method,the support may be a “gene chip” or a “biological chip” as described inU.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a genechip with polynucleotides of the present invention attached may be usedto identify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supraare hereby incorporated by reference in their entirety herein.

[0708] The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

[0709] In addition to the foregoing, a polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Antisense techniques are discussed, for example, in Okano, J.Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Triple helix formation is discussed in, for instance Lee et al., NucleicAcids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);and Dervan et al., Science 251: 1360 (1991). Both methods rely onbinding of the polynucleotide to a complementary DNA or RNA. For thesetechniques, preferred polynucleotides are usually oligonucleotides 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

[0710] The present invention encompasses the addition of a nuclearlocalization signal, operably linked to the 5′ end, 3′ end, or anylocation therein, to any of the oligonucleotides, antisenseoligonucleotides, triple helix oligonucleotides, ribozymes, PNAoligonucleotides, and/or polynucleotides, of the present invention. See,for example, G. Cutrona, et al., Nat. Biotech., 18:300-303, (2000);which is hereby incorporated herein by reference.

[0711] Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/DNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNAIDNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

[0712] The polynucleotides are also useful for identifying organismsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The polynucleotides of the presentinvention can be used as additional DNA markers for RFLP.

[0713] The polynucleotides of the present invention can also be used asan alternative to RFLP, by determining the actual base-by-base DNAsequence of selected portions of an organisms genome. These sequencescan be used to prepare PCR primers for amplifying and isolating suchselected DNA, which can then be sequenced. Using this technique,organisms can be identified because each organism will have a unique setof DNA sequences. Once an unique ID database is established for anorganism, positive identification of that organism, living or dead, canbe made from extremely small tissue samples. Similarly, polynucleotidesof the present invention can be used as polymorphic markers, in additionto, the identification of transformed or non-transformed cells and/ortissues.

[0714] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from the sequences of the present invention. Panels ofsuch reagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination. Moreover, as mentioned above, such reagents can beused to screen and/or identify transformed and non-transformed cellsand/or tissues.

[0715] In the very least, the polynucleotides of the present inventioncan be used as molecular weight markers on Southern gels, as diagnosticprobes for the presence of a specific mRNA in a particular cell type, asa probe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

[0716] Uses of the Polypeptides

[0717] Each of the polypeptides identified herein can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0718] A polypeptide of the present invention can be used to assayprotein levels in a biological sample using antibody-based techniques.For example, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0719] In addition to assaying protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0720] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131I, 112In, 99mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).)

[0721] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression of a polypeptide of thepresent invention in cells or body fluid of an individual; (b) comparingthe level of gene expression with a standard gene expression level,whereby an increase or decrease in the assayed polypeptide geneexpression level compared to the standard expression level is indicativeof a disorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0722] Moreover, polypeptides of the present invention can be used totreat, prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor suppressor), to activate the activity of a polypeptide (e.g.,by binding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

[0723] Similarly, antibodies directed to a polypeptide of the presentinvention can also be used to treat, prevent, and/or diagnose disease.For example, administration of an antibody directed to a polypeptide ofthe present invention can bind and reduce overproduction of thepolypeptide. Similarly, administration of an antibody can activate thepolypeptide, such as by binding to a polypeptide bound to a membrane(receptor).

[0724] At the very least, the polypeptides of the present invention canbe used as molecular weight markers on SDS-PAGE gels or on molecularsieve gel filtration columns using methods well known to those of skillin the art. Polypeptides can also be used to raise antibodies, which inturn are used to measure protein expression from a recombinant cell, asa way of assessing transformation of the host cell. Moreover, thepolypeptides of the present invention can be used to test the followingbiological activities.

[0725] Gene Therapy Methods

[0726] Another aspect of the present invention is to gene therapymethods for treating or preventing disorders, diseases and conditions.The gene therapy methods relate to the introduction of nucleic acid(DNA, RNA and antisense DNA or RNA) sequences into an animal to achieveexpression of a polypeptide of the present invention. This methodrequires a polynucleotide which codes for a polypeptide of the inventionthat operatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

[0727] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-I112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255(1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

[0728] As discussed in more detail below, the polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). Thepolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0729] In one embodiment, the polynucleotide of the invention isdelivered as a naked polynucleotide. The term “naked” polynucleotide,DNA or RNA refers to sequences that are free from any delivery vehiclethat acts to assist, promote or facilitate entry into the cell,including viral sequences, viral particles, liposome formulations,lipofectin or precipitating agents and the like. However, thepolynucleotides of the invention can also be delivered in liposomeformulations and lipofectin formulations and the like can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

[0730] The polynucleotide vector constructs of the invention used in thegene therapy method are preferably constructs that will not integrateinto the host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTIand pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0731] Any strong promoter known to those skilled in the art can be usedfor driving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

[0732] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0733] The polynucleotide construct of the invention can be delivered tothe interstitial space of tissues within the an animal, including ofmuscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart,lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0734] For the naked nucleic acid sequence injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05mg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration.

[0735] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0736] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0737] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0738] In certain embodiments, the polynucleotide constructs of theinvention are complexed in a liposome preparation. Liposomalpreparations for use in the instant invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. However, cationic liposomes are particularly preferredbecause a tight charge complex can be formed between the cationicliposome and the polyanionic nucleic acid. Cationic liposomes have beenshown to mediate intracellular delivery of plasmid DNA (Feigner et al.,Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is hereinincorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci.USA , 86:6077-6081 (1989), which is herein incorporated by reference);and purified transcription factors (Debs et al., J. Biol. Chem.,265:10189-10192 (1990), which is herein incorporated by reference), infunctional form.

[0739] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0740] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Felgner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0741] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0742] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0743] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca2+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA , 76:145 (1979)); and reverse-phase evaporation (REV) (Fraleyet al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl.Acad. Sci. USA , 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

[0744] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0745] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication NO: WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[0746] In certain embodiments, cells are engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding polypeptides of the invention. Retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus,and mammary tumor virus.

[0747] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy , 1:5-14 (1990), which is incorporated herein by reference inits entirety. The vector may transduce the packaging cells through anymeans known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO4 precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0748] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding polypeptides of theinvention. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express polypeptides of the invention.

[0749] In certain other embodiments, cells are engineered, ex vivo or invivo, with polynucleotides of the invention contained in an adenovirusvector. Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-I -antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

[0750] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell , 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature , 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the El region ofadenovirus and constitutively express Ela and Elb, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, AdS, and Ad7) are also useful in the present invention.

[0751] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: Ela, Elb, E3, E4, E2a, or L1 through L5.

[0752] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0753] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

[0754] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

[0755] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the desired endogenous polynucleotidesequence so the promoter will be operably linked to the endogenoussequence upon homologous recombination.

[0756] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0757] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0758] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

[0759] The polynucleotides encoding polypeptides of the presentinvention may be administered along with other polynucleotides encodingangiogenic proteins. Angiogenic proteins include, but are not limitedto, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C),VEGF-3 (VEGF-B), epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

[0760] Preferably, the polynucleotide encoding a polypeptide of theinvention contains a secretory signal sequence that facilitatessecretion of the protein. Typically, the signal sequence is positionedin the coding region of the polynucleotide to be expressed towards or atthe 5′ end of the coding region. The signal sequence may be homologousor heterologous to the polynucleotide of interest and may be homologousor heterologous to the cells to be transfected. Additionally, the signalsequence may be chemically synthesized using methods known in the art.

[0761] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers. (Kaneda et al., Science, 243:375 (1989)).

[0762] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[0763] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0764] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[0765] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-I1281 (1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0766] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

[0767] Biological Activities

[0768] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

[0769] Immune Activity

[0770] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of the immune system,by activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

[0771] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of hematopoieticcells. A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat or prevent those diseases, disorders,and/or conditions associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein diseases, disorders,and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

[0772] Moreover, a polynucleotides or polypeptides, or agonists orantagonists of the present invention could also be used to modulatehemostatic (the stopping of bleeding) or thrombolytic activity (clotformation). For example, by increasing hemostatic or thrombolyticactivity, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could be used to treat or prevent bloodcoagulation diseases, disorders, and/or conditions (e.g.,afibrinogenemia, factor deficiencies), blood platelet diseases,disorders, and/or conditions (e.g. thrombocytopenia), or woundsresulting from trauma, surgery, or other causes. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that can decrease hemostatic or thrombolytic activitycould be used to inhibit or dissolve clotting. These molecules could beimportant in the treatment or prevention of heart attacks (infarction),strokes, or scarring.

[0773] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be useful in treating, preventing, and/ordiagnosing autoimmune diseases, disorders, and/or conditions. Manyautoimmune diseases, disorders, and/or conditions result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune diseases, disorders, and/orconditions.

[0774] Examples of autoimmune diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed or detected by thepresent invention include, but are not limited to: Addison's Disease,hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis,dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves′ Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

[0775] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

[0776] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be used to treat, prevent, and/ordiagnose organ rejection or graft-versus-host disease (GVHD). Organrejection occurs by host immune cell destruction of the transplantedtissue through an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of a polynucleotidesor polypeptides, or agonists or antagonists of the present inventionthat inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

[0777] Similarly, a polynucleotides or polypeptides, or agonists orantagonists of the present invention may also be used to modulateinflammation. For example, the polypeptide or polynucleotide or agonistsor antagonist may inhibit the proliferation and differentiation of cellsinvolved in an inflammatory response. These molecules can be used totreat, prevent, and/or diagnose inflammatory conditions, both chronicand acute conditions, including chronic prostatitis, granulomatousprostatitis and malacoplakia, inflammation associated with infection(e.g., septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL- 1.)

[0778] Hyperproliferative Disorders

[0779] A polynucleotides or polypeptides, or agonists or antagonists ofthe invention can be used to treat, prevent, and/or diagnosehyperproliferative diseases, disorders, and/or conditions, includingneoplasms. A polynucleotides or polypeptides, or agonists or antagonistsof the present invention may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

[0780] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

[0781] Examples of hyperproliferative diseases, disorders, and/orconditions that can be treated, prevented, and/or diagnosed bypolynucleotides or polypeptides, or agonists or antagonists of thepresent invention include, but are not limited to neoplasms located inthe: colon, abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

[0782] Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention. Examples of such hyperproliferative diseases,disorders, and/or conditions include, but are not limited to:hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

[0783] One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

[0784] Thus, the present invention provides a method for treating orpreventing cell proliferative diseases, disorders, and/or conditions byinserting into an abnormally proliferating cell a polynucleotide of thepresent invention, wherein said polynucleotide represses saidexpression.

[0785] Another embodiment of the present invention provides a method oftreating or preventing cell-proliferative diseases, disorders, and/orconditions in individuals comprising administration of one or moreactive gene copies of the present invention to an abnormallyproliferating cell or cells. In a preferred embodiment, polynucleotidesof the present invention is a DNA construct comprising a recombinantexpression vector effective in expressing a DNA sequence encoding saidpolynucleotides. In another preferred embodiment of the presentinvention, the DNA construct encoding the polynucleotides of the presentinvention is inserted into cells to be treated utilizing a retrovirus,or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS1999 96: 324-326, which is hereby incorporated by reference). In a mostpreferred embodiment, the viral vector is defective and will nottransform non-proliferating cells, only proliferating cells. Moreover,in a preferred embodiment, the polynucleotides of the present inventioninserted into proliferating cells either alone, or in combination withor fused to other polynucleotides, can then be modulated via an externalstimulus (i.e. magnetic, specific small molecule, chemical, or drugadministration, etc.), which acts upon the promoter upstream of saidpolynucleotides to induce expression of the encoded protein product. Assuch the beneficial therapeutic affect of the present invention may beexpressly modulated (i.e. to increase, decrease, or inhibit expressionof the present invention) based upon said external stimulus.

[0786] Polynucleotides of the present invention may be useful inrepressing expression of oncogenic genes or antigens. By “repressingexpression of the oncogenic genes” is intended the suppression of thetranscription of the gene, the degradation of the gene transcript(pre-message RNA), the inhibition of splicing, the destruction of themessenger RNA, the prevention of the post-translational modifications ofthe protein, the destruction of the protein, or the inhibition of thenormal function of the protein.

[0787] For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

[0788] The polynucleotides of the present invention may be delivereddirectly to cell proliferative disorder/disease sites in internalorgans, body cavities and the like by use of imaging devices used toguide an injecting needle directly to the disease site. Thepolynucleotides of the present invention may also be administered todisease sites at the time of surgical intervention.

[0789] By “cell proliferative disease” is meant any human or animaldisease or disorder, affecting any one or any combination of organs,cavities, or body parts, which is characterized by single or multiplelocal abnormal proliferations of cells, groups of cells, or tissues,whether benign or malignant.

[0790] Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

[0791] The present invention is further directed to antibody-basedtherapies which involve administering of anti-polypeptides andanti-polynucleotide antibodies to a mammalian, preferably human, patientfor treating, preventing, and/or diagnosing one or more of the describeddiseases, disorders, and/or conditions. Methods for producinganti-polypeptides and anti-polynucleotide antibodies polyclonal andmonoclonal antibodies are described in detail elsewhere herein. Suchantibodies may be provided in pharmaceutically acceptable compositionsas known in the art or as described herein.

[0792] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0793] In particular, the antibodies, fragments and derivatives of thepresent invention are useful for treating, preventing, and/or diagnosinga subject having or developing cell proliferative and/or differentiationdiseases, disorders, and/or conditions as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

[0794] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

[0795] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of diseases, disorders,and/or conditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-6M,10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-lOM, 1O-IOM,5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M,5×10-15M, and 10-15M.

[0796] Moreover, polypeptides of the present invention may be useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

[0797] Polypeptides, including protein fusions, of the presentinvention, or fragments thereof may be useful in inhibitingproliferative cells or tissues through the induction of apoptosis. Saidpolypeptides may act either directly, or indirectly to induce apoptosisof proliferative cells and tissues, for example in the activation of adeath-domain receptor, such as tumor necrosis factor (TNF) receptor-1,CD95 (Fas/APO-1 ), TNF-receptor-related apoptosis-mediated protein(TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and-2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998),which is hereby incorporated by reference). Moreover, in anotherpreferred embodiment of the present invention, said polypeptides mayinduce apoptosis through other mechanisms, such as in the activation ofother proteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55(1998), Med Hypotheses.50(5):423-33 (1998), Chem. Biol. Interact. Apr24;111-I12:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int. J. TissueReact. 20(1):3-15 (1998), which are all hereby incorporated byreference).

[0798] Polypeptides, including protein fusions to, or fragments thereof,of the present invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231 :125-41, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

[0799] In another embodiment, the invention provides a method ofdelivering compositions containing the polypeptides of the invention(e.g., compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

[0800] Polypeptides, protein fusions to, or fragments thereof, of thepresent invention are useful in enhancing the immunogenicity and/orantigenicity of proliferating cells or tissues, either directly, such aswould occur if the polypeptides of the present invention ‘vaccinated’the immune response to respond to proliferative antigens and immunogens,or indirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

[0801] Diseases at the Cellular Level

[0802] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated, prevented, and/ordiagnosed by the polynucleotides or polypeptides and/or antagonists oragonists of the invention, include cancers (such as follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune diseases, disorders, and/orconditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, the polynucleotides or polypeptides, and/oragonists or antagonists of the invention are used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

[0803] Additional diseases or conditions associated with increased cellsurvival that could be treated, prevented or diagnosed by thepolynucleotides or polypeptides, or agonists or antagonists of theinvention, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0804] Diseases associated with increased apoptosis that could betreated, prevented, and/or diagnosed by the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, includeAIDS; neurodegenerative diseases, disorders, and/or conditions (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune diseases, disorders, and/or conditions(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

[0805] Wound Healing and Epithelial Cell Proliferation

[0806] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe invention, may be clinically useful in stimulating wound healingincluding surgical wounds, excisional wounds, deep wounds involvingdamage of the dermis and epidermis, eye tissue wounds, dental tissuewounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitusulcers, arterial ulcers, venous stasis ulcers, burns resulting from heatexposure or chemicals, and other abnormal wound healing conditions suchas uremia, malnutrition, vitamin deficiencies and complicationsassociated with systemic treatment with steroids, radiation therapy andantineoplastic drugs and antimetabolites. Polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to promote dermal reestablishment subsequent to dermal loss Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to increase the adherence of skin grafts to awound bed and to stimulate re-epithelialization from the wound bed. Thefollowing are a non-exhaustive list of grafts that polynucleotides orpolypeptides, agonists or antagonists of the invention, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepidermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, can be used to promote skin strength and to improve theappearance of aged skin.

[0807] It is believed that the polynucleotides or polypeptides, and/oragonists or antagonists of the invention, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intestine, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

[0808] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could also be used to reduce the sideeffects of gut toxicity that result from radiation, chemotherapytreatments or viral infections. The polynucleotides or polypeptides,and/or agonists or antagonists of the invention, may have acytoprotective effect on the small intestine mucosa. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, mayalso stimulate healing of mucositis (mouth ulcers) that result fromchemotherapy and viral infections.

[0809] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could further be used in full regenerationof skin in full and partial thickness skin defects, including burns,(i.e., repopulation of hair follicles, sweat glands, and sebaceousglands), treatment of other skin defects such as psoriasis. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could also be used to treat gastric anddoudenal ulcers and help heal by scar formation of the mucosal liningand regeneration of glandular mucosa and duodenal mucosal lining morerapidly. Inflamamatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to promote the resurfacing of the mucosalsurface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, isexpected to have a significant effect on the production of mucusthroughout the gastrointestinal tract and could be used to protect theintestinal mucosa from injurious substances that are ingested orfollowing surgery. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used to treat diseasesassociate with the under expression of the polynucleotides of theinvention.

[0810] Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and bums, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treated,prevented, and/or diagnosed using the polynucleotides or polypeptides,and/or agonists or antagonists of the invention. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

[0811] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

[0812] In addition, the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used treat or prevent theonset of diabetes mellitus. In patients with newly diagnosed Types I andII diabetes, where some islet cell function remains, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to maintain the islet function so as to alleviate, delay orprevent permanent manifestation of the disease. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used as an auxiliary in islet cell transplantationto improve or promote islet cell function.

[0813] Infectious Disease

[0814] A polypeptide or polynucleotide and/or agonist or antagonist ofthe present invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

[0815] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated, prevented, and/or diagnosed bya polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

[0816] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention include, but not limited to, include, but not limitedto, the following Gram-Negative and Gram-positive bacteria and bacterialfamilies and fungi: Actinomycetales (e.g., Corynebacterium,Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis,Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis,Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis,Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,Denmatocycoses, E. coli (e.g., Enterotoxigenic E. coli andEnterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella(e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia),Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria,Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

[0817] Moreover, parasitic agents causing disease or symptoms that canbe treated, prevented, and/or diagnosed by a polynucleotide orpolypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, the following families or class: Amebiasis,Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine,Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g.,Plasmodium virax, Plasmodium falciparium, Plasmodium malariae andPlasmodium ovale). These parasites can cause a variety of diseases orsymptoms, including, but not limited to: Scabies, Trombiculiasis, eyeinfections, intestinal disease (e.g., dysentery giardiasis), liverdisease, lung disease, opportunistic infections (e.g., AIDS related),malaria, pregnancy complications, and toxoplasmosis. polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedtotreat, prevent, and/or diagnose any of these symptoms or diseases. Inspecific embodiments, polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnosemalaria.

[0818] Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

[0819] Regeneration

[0820] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention can be used to differentiate, proliferate, andattract cells, leading to the regeneration of tissues. (See, Science276:59-87 (1997).) The regeneration of tissues could be used to repair,replace, or protect tissue damaged by congenital defects, trauma(wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,osteocarthritis, periodontal disease, liver failure), surgery, includingcosmetic plastic surgery, fibrosis, reperfusion injury, or systemiccytokine damage.

[0821] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0822] Moreover, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase regeneration of tissuesdifficult to heal. For example, increased tendon/ligament regenerationwould quicken recovery time after damage. A polynucleotide orpolypeptide and/or agonist or antagonist of the present invention couldalso be used prophylactically in an effort to avoid damage. Specificdiseases that could be treated, prevented, and/or diagnosed include oftendinitis, carpal tunnel syndrome, and other tendon or ligamentdefects. A further example of tissue regeneration of non-healing woundsincludes pressure ulcers, ulcers associated with vascular insufficiency,surgical, and traumatic wounds.

[0823] Similarly, nerve and brain tissue could also be regenerated byusing a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention to proliferate and differentiate nerve cells.Diseases that could be treated, prevented, and/or diagnosed using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic diseases, disorders, and/orconditions (e.g., spinal cord disorders, head trauma, cerebrovasculardisease, and stoke). Specifically, diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated, prevented, and/or diagnosed using thepolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention.

[0824] Chemotaxis

[0825] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

[0826] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase chemotaxic activity of particularcells. These chemotactic molecules can then be used to treat, prevent,and/or diagnose inflammation, infection, hyperproliferative diseases,disorders, and/or conditions, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat, prevent,and/or diagnose wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treat,prevent, and/or diagnose wounds.

[0827] It is also contemplated that a polynucleotide or polypeptideand/or agonist or antagonist of the present invention may inhibitchemotactic activity. These molecules could also be used to treat,prevent, and/or diagnose diseases, disorders, and/or conditions. Thus, apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention could be used as an inhibitor of chemotaxis.

[0828] Binding Activity

[0829] A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors),orsmall molecules.

[0830] Preferably, the molecule is closely related to the natural ligandof the polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic. (See, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which the polypeptide binds, or at least, a fragment of the receptorcapable of being bound by the polypeptide (e.g., active site). In eithercase, the molecule can be rationally designed using known techniques.

[0831] Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

[0832] The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

[0833] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

[0834] Preferably, an ELISA assay can measure polypeptide level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure polypeptide level oractivity by either binding, directly or indirectly, to the polypeptideor by competing with the polypeptide for a substrate.

[0835] Additionally, the receptor to which a polypeptide of theinvention binds can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Forexample, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

[0836] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0837] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0838] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[0839] Other preferred fragments are biologically active fragments ofthe polypeptides of the invention. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

[0840] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and 3[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0841] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the receptor is measured and the ability ofthe compound to bind to the receptor and elicit a second messengerresponse is measured to determine if the compound is a potential agonistor antagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

[0842] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

[0843] Also, one could identify molecules bind a polypeptide of theinvention experimentally by using the beta-pleated sheet regionscontained in the polypeptide sequence of the protein. Accordingly,specific embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, theamino acid sequence of each beta pleated sheet regions in a disclosedpolypeptide sequence. Additional embodiments of the invention aredirected to polynucleotides encoding polypeptides which comprise, oralternatively consist of, any combination or all of contained in thepolypeptide sequences of the invention. Additional preferred embodimentsof the invention are directed to polypeptides which comprise, oralternatively consist of, the amino acid sequence of each of the betapleated sheet regions in one of the polypeptide sequences of theinvention. Additional embodiments of the invention are directed topolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions in one of thepolypeptide sequences of the invention.

[0844] Targeted Delivery

[0845] In another embodiment, the invention provides a method ofdelivering compositions to targeted cells expressing a receptor for apolypeptide of the invention, or cells expressing a cell bound form of apolypeptide of the invention.

[0846] As discussed herein, polypeptides or antibodies of the inventionmay be associated with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions. In one embodiment, the invention provides amethod for the specific delivery of compositions of the invention tocells by administering polypeptides of the invention (includingantibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

[0847] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

[0848] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

[0849] Drug Screening

[0850] Further contemplated is the use of the polypeptides of thepresent invention, or the polynucleotides encoding these polypeptides,to screen for molecules which modify the activities of the polypeptidesof the present invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

[0851] This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

[0852] Thus, the present invention provides methods of screening fordrugs or any other agents which affect activities mediated by thepolypeptides of the present invention. These methods comprise contactingsuch an agent with a polypeptide of the present invention or a fragmentthereof and assaying for the presence of a complex between the agent andthe polypeptide or a fragment thereof, by methods well known in the art.In such a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

[0853] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to thepolypeptides of the present invention, and is described in great detailin European Patent Application 84/03564, published on Sep. 13, 1984,which is incorporated herein by reference herein. Briefly stated, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with polypeptides of the present inventionand washed. Bound polypeptides are then detected by methods well knownin the art. Purified polypeptides are coated directly onto plates foruse in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

[0854] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptides of the present invention specifically compete with a testcompound for binding to the polypeptides or fragments thereof. In thismanner, the antibodies are used to detect the presence of any peptidewhich shares one or more antigenic epitopes with a polypeptide of theinvention.

[0855] The human TRP-PLIK2 polypeptides and/or peptides of the presentinvention, or immunogenic fragments or oligopeptides thereof, can beused for screening therapeutic drugs or compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaymay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition ofactivity of the formation of binding complexes between the ion channelprotein and the agent being tested can be measured. Thus, the presentinvention provides a method for screening or assessing a plurality ofcompounds for their specific binding affinity with a TRP-PLIK2polypeptide, or a bindable peptide fragment, of this invention,comprising providing a plurality of compounds, combining the TRP-PLIK2polypeptide, or a bindable peptide fragment, with each of a plurality ofcompounds for a time sufficient to allow binding under suitableconditions and detecting binding of the TRP-PLIK2 polypeptide or peptideto each of the plurality of test compounds, thereby identifying thecompounds that specifically bind to the TRP-PLIK2 polypeptide orpeptide.

[0856] Methods of identifying compounds that modulate the activity ofthe novel human TRP-PLIK2 polypeptides and/or peptides are provided bythe present invention and comprise combining a potential or candidatecompound or drug modulator of immunoglobulin biological activity with anTRP-PLIK2 polypeptide or peptide, for example, the TRP-PLIK2 amino acidsequence as set forth in SEQ ID NOS:2, 4, 6, or 8, and measuring aneffect of the candidate compound or drug modulator on the biologicalactivity of the TRP-PLIK2 polypeptide or peptide. Such measurableeffects include, for example, physical binding interaction; the abilityto cleave a suitable immunoglobulin substrate; effects on native andcloned TRP-PLIK2-expressing cell line; and effects of modulators orother immunoglobulin-mediated physiological measures.

[0857] Another method of identifying compounds that modulate thebiological activity of the novel TRP-PLIK2 polypeptides of the presentinvention comprises combining a potential or candidate compound or drugmodulator of a immunoglobulin biological activity with a host cell thatexpresses the TRP-PLIK2 polypeptide and measuring an effect of thecandidate compound or drug modulator on the biological activity of theTRP-PLIK2 polypeptide. The host cell can also be capable of beinginduced to express the TRP-PLIK2 polypeptide, e.g., via inducibleexpression. Physiological effects of a given modulator candidate on theTRP-PLIK2 polypeptide can also be measured. Thus, cellular assays forparticular immunoglobulin modulators may be either direct measurement orquantification of the physical biological activity of the TRP-PLIK2polypeptide, or they may be measurement or quantification of aphysiological effect. Such methods preferably employ a TRP-PLIK2polypeptide as described herein, or an overexpressed recombinantTRP-PLIK2 polypeptide in suitable host cells containing an expressionvector as described herein, wherein the TRP-PLIK2 polypeptide isexpressed, overexpressed, or undergoes upregulated expression.

[0858] Another aspect of the present invention embraces a method ofscreening for a compound that is capable of modulating the biologicalactivity of a TRP-PLIK2 polypeptide, comprising providing a host cellcontaining an expression vector harboring a nucleic acid sequenceencoding a TRP-PLIK2 polypeptide, or a functional peptide or portionthereof (e.g., SEQ ID NOS:2, 4, 6, or 8); determining the biologicalactivity of the expressed TRP-PLIK2 polypeptide in the absence of amodulator compound; contacting the cell with the modulator compound anddetermining the biological activity of the expressed TRP-PLIK2polypeptide in the presence of the modulator compound. In such a method,a difference between the activity of the TRP-PLIK2 polypeptide in thepresence of the modulator compound and in the absence of the modulatorcompound indicates a modulating effect of the compound.

[0859] Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as immunoglobulin modulators can be any small chemicalcompound, or biological entity (e.g., protein, sugar, nucleic acid,lipid). Test compounds will typically be small chemical molecules andpeptides. Generally, the compounds used as potential modulators can bedissolved in aqueous or organic (e.g., DMSO-based) solutions. The assaysare designed to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source. Assays aretypically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

[0860] High throughput screening methodologies are particularlyenvisioned for the detection of modulators of the novel TRP-PLIK2polynucleotides and polypeptides described herein. Such high throughputscreening methods typically involve providing a combinatorial chemicalor peptide library containing a large number of potential therapeuticcompounds (e.g., ligand or modulator compounds). Such combinatorialchemical libraries or ligand libraries are then screened in one or moreassays to identify those library members (e.g., particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds so identified can serve as conventional lead compounds, orcan themselves be used as potential or actual therapeutics.

[0861] A combinatorial chemical library is a collection of diversechemical compounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

[0862] The preparation and screening of combinatorial chemical librariesis well known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

[0863] Devices for the preparation of combinatorial libraries arecommercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A AppliedBiosystems, Foster City, CA; 9050 Plus, Millipore, Bedford, Mass.). Inaddition, a large number of combinatorial libraries are commerciallyavailable (e.g., ComGenex, Princeton, NJ; Asinex,- Moscow, Russia;Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, MD, and thelike).

[0864] In one embodiment, the invention provides solid phase based invitro assays in a high throughput format, where the cell or tissueexpressing an ion channel is attached to a solid phase substrate. Insuch high throughput assays, it is possible to screen up to severalthousand different modulators or ligands in a single day. In particular,each well of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

[0865] In another of its aspects, the present invention encompassesscreening and small molecule (e.g., drug) detection assays which involvethe detection or identification of small molecules that can bind to agiven protein, i.e., a TRP-PLIK2 polypeptide or peptide. Particularlypreferred are assays suitable for high throughput screeningmethodologies.

[0866] In such binding-based detection, identification, or screeningassays, a functional assay is not typically required. All that is neededis a target protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

[0867] An example of such an assay is the fluorescence based thermalshift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

[0868] To purify a TRP-PLIK2 polypeptide or peptide to measure abiological binding or ligand binding activity, the source may be a wholecell lysate that can be prepared by successive freeze-thaw cycles (e.g.,one to three) in the presence of standard protease inhibitors. TheTRP-PLIK2 polypeptide may be partially or completely purified bystandard protein purification methods, e.g., affinity chromatographyusing specific antibody described infra, or by ligands specific for anepitope tag engineered into the recombinant TRP-PLIK2 polypeptidemolecule, also as described herein. Binding activity can then bemeasured as described.

[0869] Compounds which are identified according to the methods providedherein, and which modulate or regulate the biological activity orphysiology of the TRP-PLIK2 polypeptides according to the presentinvention are a preferred embodiment of this invention. It iscontemplated that such modulatory compounds may be employed in treatmentand therapeutic methods for treating a condition that is mediated by thenovel TRP-PLIK2 polypeptides by administering to an individual in needof such treatment a therapeutically effective amount of the compoundidentified by the methods described herein.

[0870] In addition, the present invention provides methods for treatingan individual in need of such treatment for a disease, disorder, orcondition that is mediated by the TRP-PLIK2 polypeptides of theinvention, comprising administering to the individual a therapeuticallyeffective amount of the TRP-PLIK2-modulating compound identified by amethod provided herein.

[0871] Antisense And Ribozyme (Antagonists)

[0872] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresponding to the sequences contained inSEQ ID NO:1, 3, 5, 7, and/or 97, or the complementary strand thereof,and/or to nucleotide sequences contained a deposited clone. In oneembodiment, antisense sequence is generated internally by the organism,in another embodiment, the antisense sequence is separately administered(see, for example, O° Connor, Neurochem., 56:560 (1991).Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Triple helix formation is discussed in, for instance, Lee et al.,Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456(1988); and Dervan et al., Science, 251:1300 (1991). The methods arebased on binding of a polynucleotide to a complementary DNA or RNA.

[0873] For example, the use of c-myc and c-myb antisense RNA constructsto inhibit the growth of the non-lymphocytic leukemia cell line HL-60and other cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2×ligation buffer (20 mM TRIS HCl pH 7.5,lOmM MgCl2, 10OMM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

[0874] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[0875] In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

[0876] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0877] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′- non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

[0878] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO:WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al.,BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g.,Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotidemay be conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

[0879] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0880] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0881] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0882] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[0883] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

[0884] While antisense nucleotides complementary to the coding regionsequence of the invention could be used, those complementary to thetranscribed untranslated region are most preferred.

[0885] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al, Science,247:1222-1225 (1990). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

[0886] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

[0887] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[0888] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[0889] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[0890] The antagonist/agonist may also be employed to treat, prevent,and/or diagnose the diseases described herein.

[0891] Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

[0892] invention, and/or (b) a ribozyme directed to the polynucleotideof the present invention.

[0893] Biotic Associations

[0894] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability, eitherdirectly or indirectly, to initiate and/or maintain biotic associationswith other organisms. Such associations may be symbiotic, nonsymbiotic,endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. Ingeneral, a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability to form bioticassociations with any member of the fungal, bacterial, lichen,mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom,phylums, families, classes, genuses, and/or species.

[0895] The mechanism by which a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may increase the hostorganisms ability, either directly or indirectly, to initiate and/ormaintain biotic associations is variable, though may include, modulatingosmolarity to desirable levels for the symbiont, modulating pH todesirable levels for the symbiont, modulating secretions of organicacids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the increased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

[0896] In an alternative embodiment, a polynucleotide or polypeptideand/or agonist or antagonist of the present invention may decrease thehost organisms ability to form biotic associations with anotherorganism, either directly or indirectly. The mechanism by which apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may decrease the host organisms ability, eitherdirectly or indirectly, to initiate and/or maintain biotic associationswith another organism is variable, though may include, modulatingosmolarity to undesirable levels, modulating pH to undesirable levels,modulating secretions of organic acids, modulating the secretion ofspecific proteins, phenolic compounds, nutrients, or the decreasedexpression of a protein required for host-biotic organisms interactions(e.g., a receptor, ligand, etc.). Additional mechanisms are known in theart and are encompassed by the invention (see, for example, “MicrobialSignalling and Communication”, eds., R. England, G. Hobbs, N. Bainton,and D. McL. Roberts, Cambridge University Press, Cambridge, (1999);which is hereby incorporated herein by reference).

[0897] The hosts ability to maintain biotic associations with aparticular pathogen has significant implications for the overall healthand fitness of the host. For example, human hosts have symbiosis withenteric bacteria in their gastrointestinal tracts, particularly in thesmall and large intestine. In fact, bacteria counts in feces of thedistal colon often approach 10¹² per milliliter of feces. Examples ofbowel flora in the gastrointestinal tract are members of theEnterobacteriaceae, Bacteriodes, in addition to a-hemolyticstreptococci, E. coli, Bifobacteria, Anaerobic cocci, Eubacteria,Costridia, lactobacilli, and yeasts. Such bacteria, among other things,assist the host in the assimilation of nutrients by breaking down foodstuffs not typically broken down by the hosts digestive system,particularly in the hosts bowel. Therefore, increasing the hosts abilityto maintain such a biotic association would help assure proper nutritionfor the host.

[0898] Aberrations in the enteric bacterial population of mammals,particularly humans, has been associated with the following disorders:diarrhea, ileus, chronic inflammatory disease, bowel obstruction,duodenal diverticula, biliary calculous disease, and malnutrition. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantenteric flora population.

[0899] The composition of the intestinal flora, for example, is basedupon a variety of factors, which include, but are not limited to, theage, race, diet, malnutrition, gastric acidity, bile salt excretion, gutmotility, and immune mechanisms. As a result, the polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,may modulate the ability of a host to form biotic associations byaffecting, directly or indirectly, at least one or more of thesefactors.

[0900] Although the predominate intestinal flora comprises anaerobicorganisms, an underlying percentage represents aerobes (e.g., E. coli).This is significant as such aerobes rapidly become the predominateorganisms in intraabdominal infections—effectively becomingopportunistic early in infection pathogenesis. As a result, there is anintrinsic need to control aerobe populations, particularly for immunecompromised individuals.

[0901] In a preferred embodiment, a polynucleotides and polypeptides,including agonists, antagonists, and fragments thereof, are useful forinhibiting biotic associations with specific enteric symbiont organismsin an effort to control the population of such organisms.

[0902] Biotic associations occur not only in the gastrointestinal tract,but also on an in the integument. As opposed to the gastrointestinalflora, the cutaneous flora is comprised almost equally with aerobic andanaerobic organisms. Examples of cutaneous flora are members of thegram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci,micrococcus, M.sedentarius), gram-positive bacilli (e.g.,Corynebacterium species, C. minutissimum, Brevibacterium species,Propoionibacterium species, P.acnes), gram-negative bacilli (e.g.,Acinebacter species), and fungi (Pityrosporum orbiculare). Therelatively low number of flora associated with the integument is basedupon the inability of many organisms to adhere to the skin. Theorganisms referenced above have acquired this unique ability. Therefore,the polynucleotides and polypeptides of the present invention may haveuses which include modulating the population of the cutaneous flora,either directly or indirectly.

[0903] Aberrations in the cutaneous flora are associated with a numberof significant diseases and/or disorders, which include, but are notlimited to the following: impetigo, ecthyma, blistering distaldactulitis, pustules, folliculitis, cutaneous abscesses, pittedkeratolysis, trichomycosis axcillaris, dermatophytosis complex, axillaryodor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheicdermititis, and Pityrosporum folliculitis, to name a few. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantcutaneous flora population.

[0904] Additional biotic associations, including diseases and disordersassociated with the aberrant growth of such associations, are known inthe art and are encompassed by the invention. See, for example,“Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.,Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia,(1998); which is hereby incorporated herein by reference).

[0905] Pheromones

[0906] In another embodiment, a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may increase theorganisms ability to synthesize, release, and/or respond to a pheromone,either directly or indirectly. Such a pheromone may, for example, alterthe organisms behavior and/or metabolism.

[0907] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may modulate the biosynthesis and/or release ofpheromones, the organisms ability to respond to pheromones (e.g.,behaviorally, and/or metabolically), and/or the organisms ability todetect pheromones, either directly or indirectly. Preferably, any of thepheromones, and/or volatiles released from the organism, or induced, bya polynucleotide or polypeptide and/or agonist or antagonist of theinvention have behavioral effects on the organism.

[0908] For example, recent studies have shown that administration ofpicogram quantities of androstadienone, the most prominent androstenepresent on male human axillary hair and on the male axillary skin, tothe female vomeronasal organ resulted in a significant reduction ofnervousness, tension and other negative feelings in the femalerecipients (Grosser-BI, et al., Psychoneuroendocrinology, 25(3): 289-99(2000)).

[0909] Other Activities

[0910] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration, as discussed above.

[0911] The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[0912] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat, prevent, and/or diagnoseneuronal damage which occurs in certain neuronal disorders orneuro-degenerative conditions such as Alzheimer's disease, Parkinson'sdisease, and AIDS-related complex. The polypeptide of the invention mayhave the ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

[0913] The polypeptide of the invention may also be employed to maintainorgans before transplantation or for supporting cell culture of primarytissues.

[0914] The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

[0915] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also increase or decrease thedifferentiation or proliferation of embryonic stem cells, besides, asdiscussed above, hematopoietic lineage.

[0916] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

[0917] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

[0918] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one- of the following, non-limitingmeans: (i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

[0919] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

[0920] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used as a food additive orpreservative, such as to increase or decrease storage capabilities, fatcontent, lipid, protein, carbohydrate, vitamins, minerals, cofactors orother nutritional components.

[0921] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one of the following, non-limiting means:(i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

[0922] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

[0923] Also preferred is a method of treatment of an individual in needof an increased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

[0924] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

[0925] References

[0926] Caterina, M. J., Leffler, A, Malmberg, A. B., Martin, W. J.,Trafton, J, Petersen-Zeitz, K. R., Koltzenburg, M, Basbaum, A. I. &Julius, D. Impaired nociception and pain sensation in mice lacking thecapsaicin receptor. Science. 288, 306-313 (2000 ).

[0927] Caterina, M. J., Rosen, T. A., Tominaga, M., Brake, A. J. &Julius, D. A capsaicin-receptor homologue with a high threshold fornoxious heat. Nature. 398, 436-41 (1999).

[0928] Duncan, L. M., Deeds, J., Hunter, J., Shao, J., Holmgren, L. M.,Woolf, E.A., Tepper, R. I. & Shyjan, A. W. Down-regulation of the novelgene melastatin correlates with potential for melanoma metastasis.Cancer Res. 58, 1515-1520 (1998).

[0929] Freichel, M., Suh, S. H., Pfeifer, A., Schweig, U., Trost, C.,Weissgerber, P., Biel, M., Philipp, S., Freise, D., Droogmans, G.,Hofmann, F., Flockerzi, V. & Nilius, B. Lack of an endothelialstore-operated Ca2+ current impairs agonist-dependent vasorelaxation inTRP4-/- mice. Nat. Cell Biol. 3, 121-127 (2001).

[0930] Harteneck, C., Plant T. D. & Schultz, G. From worm to man: threesubfamilies of TRP channels. Trends Neurosci. 23, 159-166 (2000).

[0931] Inoue, R., Okada, T., Onoue, H., Hara, Y., Shimizu, S., Naitoh,S., Ito, Y. & Mori, Y. The transient receptor potential proteinhomologue TRP6 is the essential component of vascularalpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel. CircRes. 88, 325-332 (2001).

[0932] Liman, E. R., Corey, D. P. & Dulac, C. TRP2: a candidatetransduction channel for mammalian pheromone sensory signaling. ProcNatl Acad Sci USA. 96, 5791-5796 (1999).

[0933] Missiaen, L., Robberecht, W., van den Bosch, L., Callewaert, G.,Parys, J. B., Wuytack, F., Raeymaekers, L., Nilius, B., Eggermont, J. &De Smedt, H. Abnormal intracellular Ca²⁺ homeostasis and disease. CellCalcium. 28, 1-21 (2000).

[0934] Nagamine, K., Kudoh, J., Minoshima, S., Kawasaki, K., Asakawa,S., Ito F. & Shimizu, N. Molecular cloning of a novel putative Ca²⁺channel protein (TRPC7) highly expressed in brain. Genomics 54, 124-131(1998)

[0935] Peng, J. B., Chen, X. Z., Berger, U. V., Vassilev, P. M.,Tsukaguchi, H., Brown, E. M. & Hediger, M. A. Molecular cloning andcharacterization of a channel-like transporter mediating intestinalcalcium absorption. J. Biol. Chem. 274, 22739-22746 (1999).

[0936] Prawitt, D., Enklaar, T., Klemm, G., Gartner, B., Spangenberg,C., Winterpacht, A., Higgins, M., Pelletier, J. & Zabel, B.Identification and characterization of MTR1, a novel gene with homologyto melastatin (MLSN1) and the trp family located in the BWS-WT2 criticalregion on chromosome 11p15.5 and showing allele-specific expression. HumMol Genet. 9, 203-16 (2000).

[0937] Runnels, L. W., Yue, L. & Clapham, D. E. TRP-PLIK, a bifunctionalprotein kinase and ion channel activities. Science 291, 1043-1047(2001).

[0938] Ryazanov, A. G., Ward, M. D., Mendola, C. E., Pavur, K. S.,Dorovkov, M. V., Wiedmann, M., Erdjument-Bromage, H., Tempst, P.,Parmer, T. G., Prostko, C. R., Germino, F. J. & Hait, W. N.Identification of a new class of protein kinases represented byeukaryotic elongation factor-2 kinase. Proc. Natl. Acad. Sci. USA 94,4884-4889 (1997).

[0939] Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G. &Plant, T. D. OTRPC4, a nonselective cation channel that conferssensitivity to extracellular osmolarity. Nat. Cell Biol. 2, 695-702(2000).

[0940] Tsavaler, L., Shapero, M. H., Morkowski, S. & Laus R. TRP-P8, anovel prostate-specific gene, is up-regulated in prostate cancer andother malignancies and shares high homology with transient receptorpotential calcium channel proteins. Cancer Res. 61, 3760-3769 (2001).

[0941] Walker, R. G., Willingham, A. T. & Zuker, C. S. A Drosophilamechanosensory transduction channel. Science. 287, 2229-34 (2000).

[0942] Xu, S. Z. & Beech, D. J. TrpC1 is a membrane-spanning subunit ofstore-operated Ca2+channels in native vascular smooth muscle. Circ Res.88, 84-7 (2001).

[0943] Yue, L., Peng, J. B., Hediger, M. A., Clapham, D. E. CaT1manifests the pore properties of the calcium-release-activated calciumchannel. Nature. 410, 705-709 (2001).

[0944] Zygmunt, P. M., Petersson, J., Andersson, D. A., Chuang, H.,Sorgard, M., Di Marzo V., Julius, D. & Hogestatt, E. D. Vanilloidreceptors on sensory nerves mediate the vasodilator action ofanandamide. Nature. 400, 452-457 (1999).

EXAMPLES

[0945] Description of the Preferred Embodiments

Example 1

[0946] Method Used to Identify the Novel TRP-PLIK2 Polynucleotide of thePresent Invention—Bioinformatics Analysis

[0947] Ion channel sequences (mouse TRP1a, gil1911245, SEQ ID NO:108;mouse TRP2, gil4324938, SEQ ID NO:107; mouse TRP4, gil4200415, SEQ IDNO:106; mouse TRP5, gil6048344, SEQ ID NO:105; human TRP7, gil13928756,SEQ ID NO:104; mouse TRP3, g116014703, SEQ ID NO:103; mouse TRP6,gil2979524, SEQ ID NO:102; mouse TRP8, gil5326854, SEQ ID NO:101;Drosophila NOMPC, gil7328583, SEQ ID NO:100; C.elegans Y71A12B.4,gil1065673, SEQ ID NO:99) in the TRP/NOMPC family were used as probes tosearch the human genomic sequence database. The search program used wasthe gapped BLAST program TBLASTN (Altschul et al., 1997). A multiplesequence alignment of TRP/NOMPC family members was generated using theAlign program in software Vector NTI 5.5, using the ClustalW algorithm.A Hidden Markov Model (HMM) specific for NOMPC family was constructedusing the HMMERBUILD program in the Genewise/Wise2 package from theabove multiple sequence alignment (Bateman et al., 2000). This HMM modelwas then used to search the human genomic sequence database using thesoftware program GENEWISEDB in the Genewise/Wise2 package(http://www.sanger.ac.uk/Software/Wise2/index.shtml). Results from theTBLASTN and GENEWISEDB searches were pooled and a potential TRP familymember was identified in human BAC AL354795 (Genbank Accession No.gilAL354795). The high scoring hit segments from the genomic sequencehits from BAC AL354795 were extracted and searched back againstnon-redundant protein and patent sequence databases. The most similarprotein sequence for each genomic sequence hit was used as a template topredict putative exons from the BAC AL354795 genomic sequence using theGENEWISEDB program in the Genewise/Wise2 package (FIGS. 10A-B; SEQ IDNO:10 to 24). From this analysis, exons encoding the potential novel ionchannel was identified based upon sequence homology. To extend the 5′and 3′ sequences of putative novel ion channel molecules, the genomicregions surrounding the matching exons were analyzed using GENSCAN andFGENESH programs to generate de novo exons. Based on this analysis, thepartial sequence of a novel human ion channel related gene wasidentified directly from the genomic sequences provided in FIGS. 10A-B(SEQ ID NO:10 to 24) and is shown in FIGS. 11A-C (SEQ ID NO:97 and 98).The full-length clone was obtained using a combination of N- andC-terminal cloning protocols as described below.

Example 2

[0948] Cloning the Novel Transient Receptor Potential Channel Member,TRP-PLIK2

[0949] Probe Design

[0950] Using the predicted exon genomic sequence from BAC AL354795(FIGS. 10A-B; SEQ ID NO:0 to 24), an antisense 80 bp oligo with biotinon the 5′ end was designed with the following sequence; (SEQ ID NO:25)BAC57.80 bTTCTCAATGACACCCCAAGGAGGGATTCCAACTGTCCAGATTTTTCTCAAGGAATGAGAGGAATGGGATTTCAAGGC

[0951] Solution Hybridization and DNA Capture

[0952] One microliter of the anti-sense biotinylated oligo containingone hundred and fifty nanograms was added to six microliters (sixmicrograms) of a mixture of commercially available brain, fetal brain,heart, fetal heart, kidney and fetal kidney cDNA libraries that had beenconverted into single-stranded covalently closed circular form(generated by the in vivo fI phage super-infection method) and sevenmicroliters of 100% formamide in a 0.5 ml PCR tube. The mixture washeated in a thermal cycler to 95° C. for 2 min. Fourteen microliters of2×hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2,5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA library mixtureand incubated at 42° C. for 26 hours. Hybrids between the biotinylatedoligo and the circular cDNA were isolated by diluting the hybridizationmixture to 220 microliters in a solution containing I M NaCl, 10 mMTris-HCl pH 7.5, imM EDTA, pH 8.0 and adding 125 microliters ofstreptavidin magnetic beads. This solution was incubated at 42° C. for60 min, and mixed every 5-min to re-suspend the beads. The beads areseparated from the solution with a magnet and washed three times in 200microliters of 0.1 ×SSPE, 0.1% SDS at 45° C.

[0953] The single stranded cDNAs were release from the biotinylatedoligo/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 min. Six microliters of3 M sodium acetate was added along with 15 micrograms of glycogen andthe solution ethanol precipitated with 120 microliters of 100% ethanol.The precipitated DNA was re-suspended in 12 microliters of TE (10 mMTris-HCl, pH 8.0), lmM EDTA, pH 8.0). The single stranded cDNA wasconverted into double strands in a thermal cycler by mixing 5microliters of the captured DNA with 1.5 microliters of 10 micromolarstandard T7 primer and 1.5 microliters of 10×PCR buffer.

[0954] Sequence of the primer used to the repair single-strandedcircular DNA isolated from the primary selection. T7Sport5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO:26)

[0955] The mixture was heated to 95° C. for 20 seconds then ramped downto 59 C. At this time 15 microliters of a repair mix, that was preheatedto 70° C. was added to the DNA (Repair mix contains 4 microliters of 5mM dNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25microliters of water, and 0.25 microliters of Taq polymerase). Thesolution was ramped back to 73° C. and incubated for 23 min. Therepaired DNA was ethanol precipitate and re-suspended in 10 microlitersof TE. Two microliters were electroporated per tube of 40 microliters ofE. coli DH12S cells. Three hundred and thirty three microliters areplated onto one 150-mm plate of LB agar plus 100 micrograms/milliliterof ampicillin. After overnight incubation at 37° C., the colonies fromall plates are harvested by scraping into 10 mIs of LB+50micrograms/milliliter of ampicillin and 2 mls of sterile glycerol.

[0956] The second round of selection was initiated by makingsingle-strand circular DNA from the primary selected library using themethod listed above. The single stranded circular cDNA was assayed forthe presence of BAC57/AL 354795 transcripts by PCR using the followingprimers BAC57.1s GTCCAAGCATGTTGGGGAT (SEQ ID NO:27) BAC57.1aTCTTTTCCAATAAGGTCTCTCTGG (SEQ ID NO:28)

[0957] The secondary selection was set up using the method describedabove. The resulting single-stranded circular DNA that was captured wasconverted into double strands using the BAC57.1a primer (SEQ ID NO:28).

[0958] The resulting double stranded DNA was electroporated into DHIOBand the resulting colonies inoculated into 96 deep well blocks. Afterovernight growth, DNA was prepared and screened for BAC 57/AL 354795sequences using the BAC57 primer pair (SEQ ID NO:27 and 28). The DNA wascut with Sal I and Not I and the inserts sized by agarose gelelectrophoresis. Those cDNA clones that were positive by PCR and mappedback to the original BAC from which the probe was designed, had theirinserts sized and two clones were chosen for DNA sequencing. Thesequence for one of the clones is presented in FIGS. 11A-C. The sequencewas determined to represent a partial sequence.

[0959] C-terminal Cloning

[0960] Using the predicted exon genomic sequence from BAC AL354795described as above, oligonucletides with the following sequences wasused to amplify fragments from the human kidney library (Clontech). Thereaction mixture in 50 ul containing 5 ul cDNA library, 0.5 mM eachprimer, 5 mM dNTPs (1.25 mM each), 5 ul of 10×PCR and 0.5 unit ofTaqPlus Precision polymerase (Stratagene). The reaction was repeated for30 cycles (94o C for 45 seconds, 55o C for 45 seconds, and 72o C for 4minutes). The amplified fragments were cloned into the sequencing vectorpCR4 Blunt-TOPO (Invitrogen) for sequence analysis. 95-NJF-CATGATTATCCTATCTAAGTCCCAGAAATC (SEQ ID NO:29) 95-3-NCCAAGGAATGAGAGGAATGGGATTT (SEQ ID NO:30) 95-3-C AAATCCCATTCCTCTCATTCCTTG(SEQ ID NO:31) 95-9-NC GTAGCCTGAAAGAAGGGTATGCTG (SEQ ID NO:32) 95-9-CCAGCATACCCTTCTTTCAGGCTAC (SEQ ID NO:33) 95-10-NCGAACATGTGACATCTCAGCTTTGC (SEQ ID NO:34) 95-10C GCAAAGCTGAGATGTCACATGTTC(SEQ ID NO:35) 95-105-NC AGGGCAGGATGGCTGGCTTGA (SEQ ID NO:36) 95-105-CTCAAGCCAGCCATCCTGCCCT (SEQ ID NO:37) 95-12-NC GCAAGTAGAATGCTTTCTCTTGGC(SEQ ID NO:38) 95-12-C GCCAAGAGAAAGCATTCTACTTGC (SEQ ID NO:39) 95-13-NCAGGGGAGCTCATTGTCATGGG (SEQ ID NO:40) 95-13-C CCCATGACAATGAGCTCCCCTC (SEQID NO:41) 95-14-NC CCAACCTGTAGACAGTTATTTCTTCTCC (SEQ ID NO:42) 95-14-CGGAGAAGAAATAACTGTCTACAGGTTGG (SEQ ID NO:43) 95-15-NCTTATAGTTGCATATCATCTTCTGGGGA (SEQ ID NO:44)

[0961] The resulting full-length encoding polynucleotide sequence forTRP-PLIK2 is shown in FIGS. 1A-G (SEQ ID NO:1).

[0962] Additional clones corresponding to the TRP-PLIK2 splice variantsof the present invention were isolated according to the above methods.The full-length polynucleotide sequence of each of the TRP-PLIK2 splicevariants are provided in FIGS. 2A-G (SEQ ID NO:4), FIGS. 3A-G (SEQ IDNO:6), and FIGS. 4A-G (SEQ ID NO:8) for TRP-PLIK2b, TRP-PLIK2c, andTRP-PLIK2d, respectively.

Example 3

[0963] Expression Profiling of Novel Human Immunoglobulin Protein,TRP-PLIK2

[0964] RT-PCR

[0965] A PCR primer pair was designed to measure the steady state levelsof the TRP-PLIK2 niRNA by quantitative RT-PCR. BAC57.1sGTCCAAGCATGTTGGGGAT (SEQ ID NO:27) BAC57.1a TCTTTTCCAATAAGGTCTCTCTGG(SEQ ID NO:28)

[0966] Briefly, first strand cDNA was made from commercially availablemRNA. The relative amount of cDNA used in each assay was determined byperforming a parallel experiment using a primer pair for a geneexpressed in equal amounts in all tissues, cyclophilin. The cyclophilinprimer pair detected small variations in the amount of cDNA in eachsample and these data were used for normalization of the data obtainedwith the primer pair for the TRP-PLIK2 transcript. The PCR data wasconverted into a relative assessment of the difference in transcriptabundance amongst the tissues tested and the data is presented in FIG.7. Transcripts corresponding to the TRP-PLIK2 transcript were found tobe highly expressed in the bone marrow, kidney and testis; significantlyin liver, and to a lesser extent in small intestine, spinal cord,prostate, uterus, lung, lymph node, stomach, heart, brain, thymus, andpancreas.

[0967] Northern Blot Analysis

[0968] Human tissue Northern blots (Clontech) were probed with an RNAprobe derived from a 507-bp fragment of TRP-PLIK2 (SEQ ID NO:1)amplified from the primer pair 95-3-C and 95-5-NC. Hybridization of theblot was performed at 68° C. in ExpressHyb (Clontech) for 6 hours, with1×10⁶ cpm/ml of p³² labeled probe. Autoradiography was performed for 1week at -70° C.

[0969] The results of the Northern hybridization are shown in FIG. 8. Asshown, Transcripts corresponding to the TRP-PLIK2 transcript were foundto be highly expressed in kidney, and to a lesser extent in brain andskeletal muscle.

Example 4

[0970] Method Of Assessing The Expression Profile Of The Novel TRP-PLIK2Polypeptides Of The Present Invention Using Expanded mRNA Tissue andCell Sources

[0971] Total RNA from tissues was isolated using the TriZol protocol(Invitrogen) and quantified by determining its absorbance at 260nM. Anassessment of the 18s and 28s ribosomal RNA bands was made by denaturinggel electrophoresis to determine RNA integrity.

[0972] The specific sequence to be measured was aligned with relatedgenes found in GenBank to identity regions of significant sequencedivergence to maximize primer and probe specificity. Gene-specificprimers and probes were designed using the ABI primer express softwareto amplify small amplicons (150 base pairs or less) to maximize thelikelihood that the primers function at 100% efficiency. Allprimer/probe sequences were searched against Public Genbank databases toensure target specificity. Primers and probes were obtained from ABI.

[0973] For TRP-PLIK2, the primer probe sequences were as follows ForwardPrimer 5′-AGAAAATACACTGCCGCTCAAGA-3′ (SEQ ID NO:267) Reverse Primer5′-GTTGGGACCGCCTTCCA-3′ (SEQ ID NO:268) TaqMan Probe5′-CCCACGACCGGCACGCCTT-3′ (SEQ ID NO:269)

[0974] DNA Contamination

[0975] To access the level of contaminating genomic DNA in the RNA, theRNA was divided into 2 aliquots and one half was treated with Rnase-freeDnase (Invitrogen). Samples from both the Dnase-treated and non-treatedwere then subjected to reverse transcription reactions with (RT+) andwithout (RT-) the presence of reverse transcriptase. TaqMan assays werecarried out with gene-specific primers (see above) and the contributionof genomic DNA to the signal detected was evaluated by comparing thethreshold cycles obtained with the RT+/RT- non-Dnase treated RNA to thaton the RT+/RT- Dnase treated RNA. The amount of signal contributed bygenomic DNA in the Dnased RT- RNA must be less that 10% of that obtainedwith Dnased RT+RNA. If not the RNA was not used in actual experiments.

[0976] Reverse Transcription Reaction and Sequence Detection

[0977] 100 ng of Dnase-treated total RNA was annealed to 2.5 μM of therespective gene-specific reverse primer in the presence of 5.5 mMMagnesium Chloride by heating the sample to 72° C. for 2 min and thencooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptaseand 500μM of each dNTP was added to the reaction and the tube wasincubated at 37° C. for 30 min. The sample was then heated to 90° C. for5 min to denature enzyme.

[0978] Quantitative sequence detection was carried out on an ABI PRISM7700 by adding to the reverse transcribed reaction 2.5μM forward andreverse primers, 500μM of each dNTP, buffer and 5U AmpliTaq Gold™. ThePCR reaction was then held at 94° C. for 12 min, followed by 40 cyclesof 94° C. for 15 sec and 60° C. for 30 sec.

[0979] Data Handling

[0980] The threshold cycle (Ct) of the lowest expressing tissue (thehighest Ct value) was used as the baseline of expression and all othertissues were expressed as the relative abundance to that tissue bycalculating the difference in Ct value between the baseline and theother tissues and using it as the exponent in 2^((ΔACt))

[0981] The expanded expression profile of the TRP-PLIK2 polypeptide, isprovided in FIG. 5 and are described elsewhere herein.

Example 5

[0982] Method of Assessing the Putative Kinase Activity of the TRP-PLIK2Polypeptide

[0983] A number of methods may be employed to assess the potetnialkinase activity of the TRP-PLIK2 polypeptides. One preferred method isdescribed below. A fusion construct is made whereby the TRP-PLIK2encoding polynucleotide is operably linked to the coding region of theHA protein. CHO-K1 or HEK-293 cells grown on 100-mm dishes aretransiently transfected with 8 pg of novel TRP-PLIK2-HA cDNA constructin the pTracer-CMV2 (Invitrogen) vector with LipofectAMINE 2000 (Gibco).Cells are harvested after 48 hours with 3 ml of RIPA buffer [50 mM Tris(pH 7.4), 150 mM NaCl, I mM EDTA, 1% IGEPAL CA-630, 0.5% (w/v)deoxycholate, 0.1% (w/v) SDS, and 10 mM iodoacetamide]. TRP-PLIK2-HA isimmunoprecipitated with mouse monoclonal immunoglobulin G2a (IgG2a) HAprobe (F-7) coupled to agarose (Santa Cruz Biotechnology). The agaroseis sedimented and washed three times with RIPA buffer, and 2×SDS samplebuffer is added. The samples may be resolved by SDS-PAGE and Westernblotting following standard methods. HA probe Y-11 antibody could be theprimary antibody (Santa Cruz Biotechnology), and horseradishperoxidase-linked antibody to rabbit Ig (Amersham Pharmacia Biotech)could be the secondary antibody. The SuperSignal West Dura substrate maybe used for chemiluminescent detection (Pierce)

[0984] For phosphorylation experiment, purified GST-kinase fusionproteins and mutants are incubated at 37° C. for 30 min in the presenceor absence of MBP as a test substrate in a 50-μl reaction. Thesereactions are performed in KIN buffer {50 mM Mops (pH 7.2), 100 mM NaCl,20 mM MgC12, 0.5 mM ATP, and 2 μCi of [-32P]ATP}. Immunokinase reactionscontaining immunopurified TRP-PLIK2-HA are incubated at 37° C. for 30min in a 50-μlreaction containing KIN buffer with 75 mMn-octyl--D-glucopyranoside. The reactions are terminated by the additionof 2×SDS sample buffer, and the proteins were resolved by SDS-PAGE andCoomassie staining for the GST-kinase experiment or by SDS-PAGE andWestern blotting for the immunokinase assay. The gels are dried, and 32Pincorporation is visualized by autoradiography for the GST-kinaseexperiment. For the immunokinase experiment, 32P incorporation may bevisualized by autoradiography of the transferred proteins onpolyvinylidene difluoride membrane (Bio-Rad) before Western blotting.

[0985] In the case of the TRP-PLIK2 polypeptide of the presentinvention, the kinase activity was demonstrated as described below.Briefly:

[0986] Bacterial Expression and Protein Purification

[0987] The TRP-PLIK2 kinase domain (amino acids 1428 to 2018 of SEQ IDNO:2) was subcloned into the bacterial expression vector pGEX-6p-2(Amersham) to generate a TRP-PLIK2-GST fusion protein. The primers usedwere as follows: 95-20-BamHI-C CGGGATCCACGATGGGAGTTGACAAGATCTC (SEQ IDNO: 270) AGCCTCC 95-15HA-Not-NC ATAGTTAGCGGCCGCTTAAGCGTAATCTGGAA (SEQ IDNO:271) CATCGTATGGGTATAGTTGCATATCATCTTCT GGG

[0988] The resulting TRP-PLIK2-GST fusion protein construct wastransformed into E. Coli B121-CodonPlus-RIL (Stratagene) and singlecolonies were picked and grown overnight. The overnight culture wasdiluted 1:50 and induced with 1 mM IPTG as the O.D. reached 0.7-1. Theculture was harvested after 3.5 hr and stored frozen.

[0989] The bacteria pellets were solubilized in urea buffer (20 mM TrispH 7, 150 mM NaCl, 8M urea, 7 mM β-mercaptoethanol) for 1.5 hr andcentrifuged at 10000×g for 30 min. The supernatant was dialyzed againstrenaturing buffer (10% glycerol, 20 mM Tris pH 7, 100 mM NaCl, 2.5 mMDTT, 0.1% Triton X-100, and protease inhibitor cocktail). Thesolubilized lysates were than purified using Glutathione Sepharose 4Bbeads (Amersham). The purified TRP-PLIK2 kinase domain was then cleavedfrom the GST fusion protein using proteolytic cleavage and the purifiedTRP-PLIK2 kinase domain was subjected to further analysis.

[0990] Kinase Assay

[0991] For phosphorylation experiments, the purified TRP-PLIK2-GSTfusion protein was incubated at 30° C. for 30 min in the presence orabsence of MBP or Histon as a test substrate in a 50-μl reaction. Thesereactions were performed in KIN buffer {50 mM Mops (pH 7.2), 100 mMNaCl, 20 mM MgC12, 0.5 mM ATP, and 2 μCi of [γ32P]ATP}. The reactionswere terminated by the addition of 2×SDS sample buffer, and the proteinswere resolved by SDS-PAGE and Coomassie staining for the GST-kinaseexperiment or by SDS-PAGE. The gels were dried, and 32P incorporationwas visualized by autoradiography for the GST-kinase experiment.

[0992] The results show that the TRP-PLIK2 kinase domain does containkinase activity as predicted (see FIG. 13). Moreover, the TRP-PLIK2kinase domain can be autophosphorylated and phosphorylate substratepolypeptides. MBP was determined to be a preferred substrate overHiston. These results are consistent with the TRP-PLIK2 polypeptiderepresenting a novel transient potential receptor protein.

Example 6

[0993] Complementary Polynucleotides

[0994] Antisense molecules or nucleic acid sequences complementary tothe TRP-PLIK2 protein-encoding sequence, or any part thereof, was usedto decrease or to inhibit the expression of naturally occurringTRP-PLIK2. Although the use of antisense or complementaryoligonucleotides comprising about 15 to 35 base-pairs is described,essentially the same procedure is used with smaller or larger nucleicacid sequence fragments. An oligonucleotide based on the coding sequenceof TRP-PLIK2 protein, as shown in FIGS. 1A-G, or as depicted in SEQ IDNO:1, for example, is used to inhibit expression of naturally occurringTRP-PLIK2. The complementary oligonucleotide is typically designed fromthe most unique 5′ sequence and is used either to inhibit transcriptionby preventing promoter binding to the coding sequence, or to inhibittranslation by preventing the ribosome from binding to the TRP-PLIK2protein-encoding transcript, among others. However, other regions mayalso be targeted.

[0995] Using an appropriate portion of a 5′ sequence of SEQ ID NO:1, aneffective antisense oligonucleotide includes any of about 15-35nucleotides spanning the region which translates into the signal or 5′coding sequence, among other regions, of the polypeptide as shown inFIGS. 1A-G (SEQ ID NO:2). Appropriate oligonucleotides were designedusing OLIGO 4.06 software and the TRP-PLIK2 protein coding sequence (SEQID NO:1). The preferred oligonucleotide is deoxynucleotide, or chimericdeoxynucleotide/ribonucleotide based and is provided below. Theoligonucleotide was synthesized using chemistry essentially as describedin U.S. Pat. No. 5,849,902; which is hereby incorporated herein byreference in its entirety. ID# Sequence 16520 CCUUGACAGUCUCCCACACUGACAG(SEQ ID NO:310)

[0996] The TRP-PLIK2 polypeptide has been shown to be involved in theregulation of mammalian NF-kB and apoptosis pathways. Subjecting cellswith an effective amount of the above antisense oligonucleotide resultedin a significant increase in IkBa expression/activity providingconvincing evidence that TRP-PLIK2 at least regulates the activityand/or expression of IkBa either directly, or indirectly. Moreover, theresults suggest that TRP-PLIK2 is involved in the negative regulation ofNF-ηB/IkBa activity and/or expression, either directly or indirectly.The IkBa assay used is described below and was based upon the analysisof IkBa activity as a downstream marker for proliferative signaltransduction events.

[0997] Transfection of Post-Quiescent A549 Cells With AntiSenseOligonucleotides

[0998] Materials needed:

[0999] A549 cells maintained in DMEM with high glucose (Gibco-BRL)supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, and1×penicillin/streptomycin.

[1000] Opti-MEM (Gibco-BRL)

[1001] Lipofectamine 2000 (Invitrogen)

[1002] Antisense oligomers (Sequitur)

[1003] Polystyrene tubes.

[1004] Tissue culture treated plates.

[1005] Quiescent cells were prepared as follows:

[1006] Day 0: 300, 000 A549 cells were seeded in a T75 tissue cultureflask in 10 ml of A549 media, and incubated in at 37° C., 5% CO₂ in ahumidified incubator for 48 hours.

[1007] Day 2: The T75 flasks were rocked to remove any loosely adherentcells, and the A549 growth media removed and replenished with 10 ml offresh A549 media. The cells were cultured for six days without changingthe media to create a quiescent cell population.

[1008] Day 8: Quiescent cells were plated in multi-well format andtransfected with antisense oligonucleotides.

[1009] A549 cells were transfected according to the following:

[1010] 1. Trypsinize T75 flask containing quiescent population of A549cells.

[1011] 2. Count the cells and seed 24-well plates with 60K quiescentA549 cells per well.

[1012] 3. Allow the cells to adhere to the tissue culture plate(approximately 4 hours).

[1013] 4. Transfect the cells with antisense and controloligonucleotides according to the following:

[1014] a. A 10×stock of lipofectamine 2000 (10 ug/ml is 10×) wasprepared, and diluted lipid was allowed to stand at RT for 15 minutes.Stock solution of lipofectamine 2000 was 1 mg/ml. 10×solution fortransfection was 10 ug/ml. To prepare 10O×solution, dilute 10 ul oflipofectamine 2000 stock per 1 ml of Opti-MEM (serum free media).

[1015] b. A 10×stock of each oligomer was prepared to be used in thetransfection. Stock solutions of oligomers were at 100 uM in 20 mMHEPES, pH 7.5. 10×concentration of oligomer was 0.25 uM. To prepare the10×solutions, dilute 2.5 ul of oligomer per 1 ml of Opti-MEM.

[1016] c. Equal volumes of the 10×lipofectamine 2000 stock and the10×oligomer solutions were mixed well, and incubated for 15 minutes atRT to allow complexation of the oligomer and lipid. The resultingmixture was 5×.

[1017] d. After the 15 minute complexation, 4 volumes of full growthmedia was added to the oligomer/lipid complexes (solution was 1×).

[1018] e. The media was aspirated from the cells, and 0.5 ml of the1×oligomer/lipid complexes added to each well.

[1019] f. The cells were incubated for 16-24 hours at 37° C. in ahumidified CO₂ incubator.

[1020] g. Cell pellets were harvested for RNA isolation and TaqMananalysis of downstream marker genes.

[1021] TaqMan Reactions

[1022] Quantitative RT-PCR analysis was performed on total RNA prepsthat had been treated with DNaseI or poly A selected RNA. The Dnasetreatment may be performed using methods known in the art, thoughpreferably using a Qiagen RNeasy kit to purify the RNA samples, whereinDNAse I treatment is performed on the column.

[1023] Briefly, a master mix of reagents was prepared according to thefollowing table: Dnase I Treatment Reagent Per r′xn (in uL) 10x Buffer2.5 Dnase I (1 unit/ul @ 1 unit per ug 2 sample) DEPC H₂O 0.5 RNA sample@ 0.1 ug/ul 20 (2-3 ug total) Total 25

[1024] Next, 5 ul of master mix was aliquoted per well of a 96-well PCRreaction plate (PE part # N801-0560). RNA samples were adjusted to 0.1ug/ul with DEPC treated H₂O (if necessary), and 20 ul was added to thealiquoted master mix for a final reaction volume of 25 ul.

[1025] The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and briefly spun in a centrifuge tocollect all volume in the bottom of the tubes. Generally, a short spinup to 500 rpm in a Sorvall RT is sufficient The plates were incubated at37° C. for 30 mins. Then, an equal volume of 0.1 mM EDTA in 10 mM Triswas added to each well, and heat inactivated at 70° C. for 5 min. Theplates were stored at −80° C. upon completion.

[1026] RT Reaction

[1027] A master mix of reagents was prepared according to the followingtable: RT reaction RT No RT Reagent Per Rx′n (in ul) Per Rx′n (in ul)10x RT buffer 5 2.5 MgCl₂ 11 5.5 DNTP mixture 10 5 Random Hexamers 2.51.25 Rnase inhibitors 1.25 0.625 RT enzyme 1.25 — Total RNA 500 ng (100ng no RT) 19.0 max 10.125 max DEPC H₂O — — Total 50 uL 25 uL

[1028] Samples were adjusted to a concentration so that 500 ng of RNAwas added to each RT rx'n (1Ong for the no RT). A maximum of 19 ul canbe added to the RT rx'n mixture (10.125 ul for the no RT.) Any remainingvolume up to the maximum values was filled with DEPC treated H₂0, sothat the total reaction volume was 50 ul (RT) or 25 ul (no RT).

[1029] On a 96-well PCR reaction plate (PE part # N801-0560), 37.5 ul ofmaster mix was aliquoted (22.5 ul of no RT master mix), and the RNAsample added for a total reaction volume of 50ul (25 ul, no RT). Controlsamples were loaded into two or even three different wells in order tohave enough template for generation of a standard curve.

[1030] The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and spin briefly in a centrifuge tocollect all volume in the bottom of the tubes. Generally, a short spinup to 500 rpm in a Sorvall RT is sufficient.

[1031] For the RT-PCR reaction, the following thermal profile was used:

[1032] 25° C. for 10 min

[1033] 48° C. for 30 min

[1034] 95° C. for 5 min

[1035] 4° C. hold (for 1 hour)

[1036] Store plate @-20° C. or lower upon completion.

[1037] TaqMan Reaction (Template Comes from RTplate.)

[1038] A master mix was prepared according to the following table:TaqMan reaction (per well) Reagent Per Rx′n (in ul) TaqMan Master Mix4.17 100 uM Probe .025 (SEQ ID NO:309) 100 uM Forward .05 primer (SEQ IDNO:307) 100 uM Reverse .05 primer (SEQ ID NO:308) Template — DEPC H₂O18.21 Total 22.5

[1039] The primers used for the RT-PCR reaction is as follows: IkBaprimer and probes: Forward Primer: GAGGATGAGGAGAGCTATGACACA (SEQ IDNO:307) Reverse Primer: CCCTTTGCACTCATAACGTCAG (SEQ ID NO:308) TaqManProbe: AAACACACAGTCATCATAGGGCAGCTCGT (SEQ ID NO:309)

[1040] Using a Gilson P-10 repeat pipetter, 22.5 ul of master mix wasaliquouted per well of a 96-well optical plate. Then, using P-10pipetter, 2.5 ul of sample was added to individual wells. Generally, RTsamples are run in triplicate with each primer/probe set used, and no RTsamples are run once and only with one primer/probe set, often gapdh (orother internal control).

[1041] A standard curve is then constructed and loaded onto the plate.The curve has five points plus one no template control (NTC,=DEPCtreated H₂O). The curve was made with a high point of 50 ng of sample(twice the amount of RNA in unknowns), and successive samples of 25, 10,5, and 1 ng. The curve was made from a control sample(s) (see above).

[1042] The wells were capped using optical strip well caps (PE part #N801-0935), placed in a plate, and spun in a centrifuge to collect allvolume in the bottom of the tubes. Generally, a short spin up to 500 rpmin a Sorvall RT is sufficient.

[1043] Plates were loaded onto a PE 5700 sequence detector making surethe plate is aligned properly with the notch in the upper right handcomer. The lid was tightened down and run using the 5700 and 5700quantitation program and the SYBR probe using the following thermalprofile:

[1044] 50° C. for 2 min

[1045] 95° C. for 10 min

[1046] and the following for 40 cycles:

[1047] 95° C. for 15 sec

[1048] 60° C. for 1 min

[1049] Change the reaction volume to 25ul.

[1050] Once the reaction was complete, a manual threshold of around 0.1was set to minimuze the background signal.Additional informationrelative to operation of the GeneAmp 5700 machine may be found inreference to the following manuals: “GeneAmp 5700 Sequence DetectionSystem Operator Training CD” ; and the “User's Manual for 5700 SequenceDetection System” ; available from Perkin-Elmer and hereby incorporatedby reference herein in their entirety.

Example 7

[1051] Method of Assessing the Putative Ion Channel Activity of theTRP-PLIK2 Polypeptide

[1052] A number of methods may be employed to assess the potential ionchannel activity of the TRP-PLIK2 polypeptides. One preferred method isdescribed below CHO-K1 cells transfected with a suitable mammalianexpression vector comprising the TRP-PLIK2 encoding polynucleotidesequence is prepared using methods known in the art. The transfectedcells are transferred to cover slips 12 hours after transfection, andelectrophysiological measurements are made 24 hours after transfection(22±2° C.). The TRP-PLIK2 -expressing CHO-K1 cells are detected by GFPfluorescence. Membrane currents are digitized at 10 or 20 kHz anddigitally filtered off line at 1 kHz. Voltage stimuli lasting 500 ms aredelivered at 5-s intervals, with either voltage ramps or voltage stepsfrom 100 to +100 mV. The internal pipette solution for macroscopic andsingle-channel currents may contain 145 mM Cs-methanesulfonate, 8 mMNaCl, 5 mM ATP, 1 mM MgCl2, 10 mM EGTA, 4.1 mM CaCl2, and 10 mM Hepes,with pH adjusted to 7.2 with CsOH after addition of ATP. The standardextracellular solution may contain 140 mM NaCl, 5 mM CsCl, 2.8 mM KCl, 2mM CaCl2, 1 mM MgCl2, 10 mM Hepes, and 10 mM glucose, with pH adjustedto 7.4 with NaOH. Relative ion permeabilities may be measured with thepipette solution containing 145 mM Cs-methanesulfonate, 10 mM CsCl, 5 mMATP, 10 mM EGTA, and 10 mM Hepes (pH 7.2) and the external solutioncontaining 110 mM NMDG+, 30 mM X+(Na+, Ca2+, K+, or Cs+), 10 mM Hepes,and 10 mM glucose (pH 7.4). The relative permeability for monovalentions may be calculated according to the equationPX/PCs=([Cs+]o/[X+]o)exp[F(EX ECs)/RT]. The PCa/PCs permeability ratiois calculated according to the equationPCa/PCs={[Cs+]oexp(FECs/RT)exp(FECa/RT)[exp(FECa/RT)+1 ]}/(4[Ca2+]o),where R, T, and F are the gas constant, absolute temperature, andFaraday's constant, respectively. Statistical comparisons are made withthe two-way analysis of ariance (ANOVA) and two-tailed t test withBonferroni correction; P<0.05 indicated statistical significance.

Example 8

[1053] Method Of Assessing Ability Of TRP-PLIK2 Polypeptides ToAssociate With Other Proteins Using The Yeast Two-Hybrid System

[1054] In an effort to determine whether the TRP-PLIK2 polypeptides ofthe present invention are capable of functioning as an ion channel orkinase protein, it would be important to effectively test theinteraction between TRP-PLIK2 and various portions of other proteins,particularly known ion channel proteins, for example, in a yeasttwo-hybrid system. Such a system could be created using methods known inthe art (see, for example, S. Fields and O. Song, Nature, 340:245-246(1989); and Gaston-SM and Loughlin-KR, Urology, 53(4): 835-42 (1999);which are hereby incorporated herein by reference in their entirety,including the articles referenced therein).

[1055] Cytoplasmic NH and COOH terminal domains of different proteins,preferably ion channel proteins (such as those referenced herein), couldbe subcloned and expressed as fusion proteins of the GAL4 DNA binding(DB) domain using molecular biology techniques within the skill of theartisan.

[1056] Exemplary subunits which could be used in the two-hybrid systemto assess TRP-PLIK2s ability to associate with other ion channelproteins include, but are not limited to, the NH and/or C-terminaldomain TRP1, TRP2, TRP3, TRP4, TRP5, TRP6, TRP7, signalling proteins,etc.

Example 9

[1057] Method Of Assessing Ability Of TRP-PLIK2 Polypeptides To FormOligomeric Complexes With Itself Or Other Ion Channel Proteins InSolution

[1058] Aside from determining whether the TRP-PLIK2 polypeptides arecapable of interacting with other proteins, preferably ion channelproteins, in a yeast two-hybrid assay, it would be an important nextstep to assess its ability to form oligomeric complexes with itself, inaddition to other proteins, preferably ion channel proteins, insolution. Such a finding would be significant as it would provideconvincing evidence that TRP-PLIK2 could serve as an ion channelprotein.

[1059] A number of methods could be used to that are known in the art,for example, the method described by Sanguinetti, M. C., et al., Nature,384:80-83 (1996) could be adapted using methods within the skill of theartisan.

Example 10

[1060] Method of Identifying the Cognate Ligand of the TRP-PLIK2Polypeptide

[1061] A number of methods are known in the art for identifying thecognate binding partner of a particular polypeptide. For example, theencoding TRP-PLIK2 polynucleotide could be engineered to comprise anepitope tag. The epitope could be any epitope known in the art ordisclosed elsewhere herein. Once created, the epitope tagged TRP-PLIK2encoding polynucleotide could be cloned into an expression vector andused to transfect a variety of cell lines representing different tissueorigins (e.g., brain, testis, kindey, testis, liver, etc.). Thetransfected cell lines could then be induced to overexpress theTRP-PLIK2 polypeptide. The presence of the TRP-PLIK2 polypeptide on thecell surface could be determined by fractionating whole cell lysatesinto cellular and membrane protein fractions and performingimmunoprecipitation using the antibody directed against the epitopeengineered into the TRP-PLIK2 polypeptide. Monoclonal or polyclonalantibodies directed against the TRP-PLIK2 polypeptide could be createdand used in place of the antibodies directed against the epitope.

[1062] Alternatively, the cell surface proteins could be distinguishedfrom cellular proteins by biotinylating the surface proteins and thenperforming immunoprecipitations with antibody specific to the TRP-PLIK2protein. After electrophoretic separation, the biotinylated proteincould be detected with streptavidin-HRP (using standard methods known tothose skilled in the art). Identification of the proteins bound toTRP-PLIK2 could be made in those cells by immunoprecipation, followed byone-dimensional electrophoresis, followed by various versions of massspectrometry. Such mass-spectrometry methods are known in the art, suchas for example the methods taught by Ciphergen Biosystems Inc. (see U.S.Pat. No. 5,792,664; which is hereby incorporated herein by reference).

Example 11

[1063] Isolation of a Specific Clone from the Deposited Sample

[1064] The deposited material in the sample assigned the ATCC DepositNumber cited in Table 1 for any given cDNA clone also may contain one ormore additional plasmids, each comprising a cDNA clone different fromthat given clone. Thus, deposits sharing the same ATCC Deposit Numbercontain at least a plasmid for each cDNA clone identified in Table 1.Typically, each ATCC deposit sample cited in Table 1 comprises a mixtureof approximately equal amounts (by weight) of about 1-10 plasmid DNAs,each containing a different cDNA clone and/or partial cDNA clone; butsuch a deposit sample may include plasmids for more or less than 2 cDNAclones.

[1065] Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNA(s) cited for that clone in Table 1.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:1, 3,5,7,and/or 97.

[1066] Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with32P-(-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1. 104), or other techniquesknown to those of skill in the art.

[1067] Alternatively, two primers of 17-20 nucleotides derived from bothends of the SEQ ID NO:1, 3, 5, 7, and/or 97 (i.e., within the region ofSEQ ID NO:1, 3, 5, 7, and/or 97 bounded by the 5′ NT and the 3′ NT ofthe clone defined in Table 1) are synthesized and used to amplify thedesired cDNA using the deposited cDNA plasmid as a template. Thepolymerase chain reaction is carried out under routine conditions, forinstance, in 25 ul of reaction mixture with 0.5 ug of the above cDNAtemplate. A convenient reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v)gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primerand 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturationat 94 degree C. for 1 min; annealing at 55 degree C. for 1 min;elongation at 72 degree C. for 1 min) are performed with a Perkin-ElmerCetus automated thermal cycler. The amplified product is analyzed byagarose gel electrophoresis and the DNA band with expected molecularweight is excised and purified. The PCR product is verified to be theselected sequence by subcloning and sequencing the DNA product.

[1068] The polynucleotide(s) of the present invention, thepolynucleotide encoding the polypeptide of the present invention, or thepolypeptide encoded by the deposited clone may represent partial, orincomplete versions of the complete coding region (i.e., full-lengthgene). Several methods are known in the art for the identification ofthe 5′ or 3′ non-coding and/or coding portions of a gene which may notbe present in the deposited clone. The methods that follow are exemplaryand should not be construed as limiting the scope of the invention.These methods include but are not limited to, filter probing, cloneenrichment using specific probes, and protocols similar or identical to5′ and 3′ “RACE” protocols that are well known in the art. For instance,a method similar to 5′ RACE is available for generating the missing 5′end of a desired full-length transcript. (Fromont-Racine et al., NucleicAcids Res. 21(7):1683-1684 (1993)).

[1069] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full-length gene.

[1070] This above method starts with total RNA isolated from the desiredsource, although poly-A+RNA can be used. The RNA preparation can then betreated with phosphatase if necessary to eliminate 5′ phosphate groupson degraded or damaged RNA that may interfere with the later RNA ligasestep. The phosphatase should then be inactivated and the RNA treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase.

[1071] This modified RNA preparation is used as a template for firststrand cDNA synthesis using a gene specific oligonucleotide. The firststrand synthesis reaction is used as a template for PCR amplification ofthe desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene. Moreover,it may be advantageous to optimize the RACE protocol to increase theprobability of isolating additional 5′ or 3′ coding or non-codingsequences. Various methods of optimizing a RACE protocol are known inthe art, though a detailed description summarizing these methods can befound in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

[1072] An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding sequences is provided by Frohman,M. A., et al., Proc.Nat'l.Acad.Sci.USA, 85:8998-9002 (1988). Briefly, acDNA clone missing either the 5′ or 3′ end can be reconstructed toinclude the absent base pairs extending to the translational start orstop codon, respectively. In some cases, cDNAs are missing the start oftranslation, therefor. The following briefly describes a modification ofthis original 5′ RACE procedure. Poly A+or total RNAs reversetranscribed with Superscript II (Gibco/BRL) and an antisense or Icomplementary primer specific to the cDNA sequence. The primer isremoved from the reaction with a Microcon Concentrator (Amicon). Thefirst-strand cDNA is then tailed with dATP and terminal deoxynucleotidetransferase (Gibco/BRL). Thus, an anchor sequence is produced which isneeded for PCR amplification. The second strand is synthesized from thedA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), anoligo-dT primer containing three adjacent restriction sites (XhoIJ Sailand ClaI) at the 5′ end and a primer containing just these restrictionsites. This double-stranded cDNA is PCR amplified for 40 cycles with thesame primers as well as a nested cDNA-specific antisense primer. The PCRproducts are size-separated on an ethidium bromide-agarose gel and theregion of gel containing cDNA products the predicted size of missingprotein-coding DNA is removed. cDNA is purified from the agarose withthe Magic PCR Prep kit (Promega), restriction digested with XhoI orSalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) atXhoI and EcoRV sites. This DNA is transformed into bacteria and theplasmid clones sequenced to identify the correct protein-coding inserts.Correct 5′ ends are confirmed by comparing this sequence with theputatively identified homologue and overlap with the partial cDNA clone.Similar methods known in the art and/or commercial kits are used toamplify and recover 3′ ends.

[1073] Several quality-controlled kits are commercially available forpurchase. Similar reagents and methods to those above are supplied inkit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of fulllength genes. A second kit is available from Clontech which is amodification of a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32(1991). The major differences in procedure are that the RNA isalkaline hydrolyzed after reverse transcription and RNA ligase is usedto join a restriction site-containing anchor primer to the first-strandcDNA. This obviates the necessity for the dA-tailing reaction whichresults in a polyT stretch that is difficult to sequence past.

[1074] An alternative to generating 5′ or 3′ cDNA from RNA is to usecDNA library double- stranded DNA. An asymmetric PCR-amplified antisensecDNA strand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

[1075] RNA Ligase Protocol For Generating The 5′ or 3′ End Sequences ToObtain Full Length Genes

[1076] Once a gene of interest is identified, several methods areavailable for the identification of the 5′ or 3′ portions of the genewhich may not be present in the original cDNA plasmid. These methodsinclude, but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar and identical to 5′ and 3'RACE.While the full-length gene may be present in the library and can beidentified by probing, a useful method for generating the 5′ or 3′ endis to use the existing sequence information from the original cDNA togenerate the missing information. A method similar to 5'RACE isavailable for generating the missing 5′ end of a desired full-lengthgene. (This method was published by Fromont-Racine et al., Nucleic AcidsRes., 21(7): 1683-1684 (1993)). Briefly, a specific RNA oligonucleotideis ligated to the 5′ ends of a population of RNA presumably 30containing full-length gene RNA transcript and a primer set containing aprimer specific to the ligated RNA oligonucleotide and a primer specificto a known sequence of the gene of interest, is used to PCR amplify the5′ portion of the desired full length gene which may then be sequencedand used to generate the full length gene. This method starts with totalRNA isolated from the desired source, poly A RNA may be used but is nota prerequisite for this procedure. The RNA preparation may then betreated with phosphatase if necessary to eliminate 5′ phosphate groupson degraded or damaged RNA which may interfere with the later RNA ligasestep. The phosphatase if used is then inactivated and the RNA is treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNApreparation can then be used as a template for first strand cDNAsynthesis using a gene specific oligonucleotide. The first strandsynthesis reaction can then be used as a template for PCR amplificationof the desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of theapoptosis related of interest. The resultant product is then sequencedand analyzed to confirm that the 5′ end sequence belongs to the relevantapoptosis related.

Example 12

[1077] Chromosomal Mapping of the Polynucleotides

[1078] An oligonucleotide primer set is designed according to thesequence at the 5′ end of SEQ ID NO:1. This primer preferably spansabout 100 nucleotides. This primer set is then used in a polymerasechain reaction under the following set of conditions: 30 seconds,95degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle isrepeated 32 times followed by one 5 minute cycle at 70 degree C.Mammalian DNA, preferably human DNA, is used as template in addition toa somatic cell hybrid panel containing individual chromosomes orchromosome fragments (Bios, Inc). The reactions are analyzed on either8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping isdetermined by the presence of an approximately 100 bp PCR fragment inthe particular somatic cell hybrid.

Example 13

[1079] Bacterial Expression of a Polypeptide

[1080] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 11, tosynthesize insertion fragments. The primers used to amplify the cDNAinsert should preferably contain restriction sites, such as BamHI andXbaI, at the 5′ end of the primers in order to clone the amplifiedproduct into the expression vector. For example, BamHI and XbaIcorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vectorencodes antibiotic resistance (Ampr), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

[1081] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, that expresses the lacIrepressor and also confers kanamycin resistance (Kanr). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[1082] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.600) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[1083] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000×g). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl by stirringfor 3-4 hours at 4 degree C. The cell debris is removed bycentrifugation, and the supernatant containing the polypeptide is loadedonto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6 ×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[1084] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[1085] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4 degree C. or frozen at −80 degree C.

Example 14

[1086] Purification of a Polypeptide from an Inclusion Body

[1087] The following alternative method can be used to purify apolypeptide expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10 degree C.

[1088] Upon completion of the production phase of the E. colifermentation, the cell culture is cooled to 4-10 degree C. and the cellsharvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech).On the basis of the expected yield of protein per unit weight of cellpaste and the amount of purified protein required, an appropriate amountof cell paste, by weight, is suspended in a buffer solution containing100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to ahomogeneous suspension using a high shear mixer.

[1089] The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[1090] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

[1091] Following high speed centrifugation (30,000×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4 degree C. without mixingfor 12 hours prior to further purification steps.

[1092] To clarify the refolded polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 urn membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 nm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

[1093] Fractions containing the polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perceptive Biosystems) and weak anion (Poros CM-20, PerceptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A280 monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

[1094] The resultant polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Coomassie blue stained 16% SDS-PAGE gelwhen 5 ug of purified protein is loaded. The purified protein can alsobe tested for endotoxin/LPS contamination, and typically the LPS contentis less than 0.1 ng/ml according to LAL assays.

Example 15

[1095] Cloning and Expression of a Polypeptide in a BaculovirusExpression System

[1096] In this example, the plasmid shuttle vector pAc373 is used toinsert a polynucleotide into a baculovirus to express a polypeptide. Atypical baculovirus expression vector contains the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV) followed by convenient restriction sites, which may include,for example BamHI, Xba I and Asp7l8. The polyadenylation site of thesimian virus 40 (“SV40”) is often used for efficient polyadenylation.For easy selection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate a viable virus that express the clonedpolynucleotide.

[1097] Many other baculovirus vectors can be used in place of the vectorabove, such as pVL941 and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

[1098] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 11, tosynthesize insertion fragments. The primers used to amplify the cDNAinsert should preferably contain restriction sites at the 5′ end of theprimers in order to clone the amplified product into the expressionvector. Specifically, the cDNA sequence contained in the depositedclone, including the AUG initiation codon and the naturally associatedleader sequence identified elsewhere herein (if applicable), isamplified using the PCR protocol described in Example 11. If thenaturally occurring signal sequence is used to produce the protein, thevector used does not need a second signal peptide. Alternatively, thevector can be modified to include a baculovirus leader sequence, usingthe standard methods described in Summers et al., “A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experimental Station Bulletin No. 1555 (1987).

[1099] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[1100] The plasmid is digested with the corresponding restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[1101] The fragment and the dephosphorylated plasmid are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria containing the plasmid are identified by digesting DNAfrom individual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

[1102] Five ug of a plasmid containing the polynucleotide isco-transformed with 1.0 ug of a commercially available linearizedbaculovirus DNA (“BaculoGoldtm baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldtm virusDNA and 5 ug of the plasmid are mixed in a sterile well of a microtiterplate containing 50 ul of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ulGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

[1103] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10.) After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

[1104] To verify the expression of the polypeptide, Sf9 cells are grownin Grace's medium supplemented with 10% heat-inactivated FBS. The cellsare infected with the recombinant baculovirus containing thepolynucleotide at a multiplicity of infection (“MOI”) of about 2. Ifradiolabeled proteins are desired, 6 hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). After 42 hours,5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[1105] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the produced protein.

Example 16

[1106] Expression of a Polypeptide in Mammalian Cells

[1107] The polypeptide of the present invention can be expressed in amammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

[1108] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, human Hela, 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CVI, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[1109] Alternatively, the polypeptide can be expressed in stable celllines containing the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

[1110] The transformed gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem. . . . 253:1357-1370 (1978); Hamlin, J. L.and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J.and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992). Using these markers, the mammalian cells are grown inselective medium and the cells with the highest resistance are selected.These cell lines contain the amplified gene(s) integrated into achromosome. Chinese hamster ovary (CHO) and NSO cells are often used forthe production of proteins.

[1111] A polynucleotide of the present invention is amplified accordingto the protocol outlined in herein. If the naturally occurring signalsequence is used to produce the protein, the vector does not need asecond signal peptide. Alternatively, if the naturally occurring signalsequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891.) The amplifiedfragment is isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). Thefragment then is digested with appropriate restriction enzymes and againpurified on a 1% agarose gel.

[1112] The amplified fragment is then digested with the same restrictionenzyme and purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB11 or XL-1 Blue cells are then transformed and bacteria are identifiedthat contain the fragment inserted into plasmid pC6 using, for instance,restriction enzyme analysis.

[1113] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transformation. Five μg of an expression plasmid is cotransformedwith 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 17

[1114] Protein Fusions

[1115] The polypeptides of the present invention are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example described herein; see also EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the half-life time in vivo.Nuclear localization signals fused to the polypeptides of the presentinvention can target the protein to a specific subcellular localization,while covalent heterodimer or homodimers can increase or decrease theactivity of a fusion protein. Fusion proteins can also create chimericmolecules having more than one function. Finally, fusion proteins canincrease solubility and/or stability of the fused protein compared tothe non-fused protein. All of the types of fusion proteins describedabove can be made by modifying the following protocol, which outlinesthe fusion of a polypeptide to an IgG molecule.

[1116] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector. Note that the polynucleotideis cloned without a stop codon, otherwise a fusion protein will not beproduced.

[1117] The naturally occurring signal sequence may be used to producethe protein (if applicable). Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat.No. 6,066,781, supra.) Human IgG Fc region: (SEQ ID NO:262)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 18

[1118] Method of Creating N- and C-terminal Deletion MutantsCorresponding to the TRP-PLIK2 Polypeptide of the Present Invention

[1119] As described elsewhere herein, the present invention encompassesthe creation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to theTRP-PLIK2 polypeptide of the present invention. A number of methods areavailable to one skilled in the art for creating such mutants. Suchmethods may include a combination of PCR amplification and gene cloningmethodology. Although one of skill in the art of molecular biology,through the use of the teachings provided or referenced herein, and/orotherwise known in the art as standard methods, could readily createeach deletion mutant of the present invention, exemplary methods aredescribed below.

[1120] Briefly, using the isolated cDNA clone encoding the full-lengthTRP-PLIK2 polypeptide sequence (as described in Example 11, forexample), appropriate primers of about 15-25 nucleotides derived fromthe desired 5′ and 3′ positions of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, or SEQ ID NO:7 may be designed to PCR amplify, and subsequentlyclone, the intended N- and/or C-terminal deletion mutant. Such primerscould comprise, for example, an inititation and stop codon for the 5′and 3′ primer, respectively. Such primers may also comprise restrictionsites to facilitate cloning of the deletion mutant post amplification.Moreover, the primers may comprise additional sequences, such as, forexample, flag-tag sequences, kozac sequences, or other sequencesdiscussed and/or referenced herein.

[1121] For example, in the case of the W739 to L2017 TRP-PLIK2N-terminal deletion mutant, the following primers could be used toamplify a cDNA fragment corresponding to this deletion mutant: 5′ Primer5′-GCAGCA GCGGCCGC TGGTTAAAGATTATTATAAGC-3′ (SEQ ID NO:263)              

                                                  3′ Primer 5′-GCAGCAGTCGAC TAGTTGCATATCATCTTCTGGGG-3′ (SEQ ID NO:264)               

                                                 

[1122] For example, in the case of the M1 to P1287 TRP-PLIK2 C-terminaldeletion mutant, the following primers could be used to amplify a cDNAfragment corresponding to this deletion mutant: 5′ Primer 5′-GCAGCACCGGCCGC ATGATTATCCTATCTAAGTCCCAG-3′ (SEQ ID NO:265)                

                                                     3′ Primer 5′-GCAGCAGTCGAC GGGATGCCGGCCTCCAGCCAGGCTC-3′ (SEQ ID NO:266)                

                                                    

[1123] The resulting C-terminal deletion mutant could be used as apotential, membrane bound, TRP-PLIK2 decoy receptor. Any C-terminaldeletion between amino acid 1200 to 1600 of SEQ ID NO:2 would beprefered.

[1124] Representative PCR amplification conditions are provided below,although the skilled artisan would appreciate that other conditions maybe required for efficient amplification. A 100 ul PCR reaction mixturemay be prepared using long of the template DNA (cDNA clone of TRP-PLIK2,TRP-PLIK2b, TRP-PLIK2c, or TRP-PLIK2d), 200 uM 4dNTPs, luM primers,0.25U Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer.Typical PCR cycling condition are as follows:

[1125] 20-25 cycles:45 sec, 93 degrees

[1126] 2 min, 50 degrees

[1127] 2 min, 72 degrees

[1128] 1 cycle: 10 min, 72 degrees

[1129] After the final extension step of PCR, 5U Klenow Fragment may beadded and incubated for 15 min at 30 degrees.

[1130] Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). . The skilled artisan would appreciate that otherplasmids could be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

[1131] The 5′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula:

[1132] (S+(X * 3)) to ((S+(X * 3))+25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the TRP-PLIK2 gene(SEQ ID NO:1), TRP-PLIK2b gene (SEQ ID NO:3), TRP-PLIK2c gene (SEQ IDNO:5), or TRP-PLIK2d gene (SEQ ID NO:7) and ‘X’ is equal to the mostN-terminal amino acid of the intended N-terminal deletion mutant. Thefirst term will provide the start 5′ nucleotide position of the 5′primer, while the second term will provide the end 3′ nucleotideposition of the 5′ primer corresponding to sense strand of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. Once the correspondingnucleotide positions of the primer are determined, the final nucleotidesequence may be created by the addition of applicable restriction sitesequences to the 5′ end of the sequence, for example. As referencedherein, the addition of other sequences to the 5′ primer may be desiredin certain circumstances (e.g., kozac sequences, etc.).

[1133] The 3′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula:

[1134] (S+(X * 3)) to ((S+(X * 3))-25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the TRP-PLIK2 gene(SEQ ID NO:1), TRP-PLIK2b gene (SEQ ID NO:3), TRP-PLIK2c gene (SEQ IDNO:5), or TRP-PLIK2b gene (SEQ ID NO:7) and ‘X’ is equal to the mostC-terminal amino acid of the intended N-terminal deletion mutant. Thefirst term will provide the start 5′ nucleotide position of the 3′primer, while the second term will provide the end 3′ nucleotideposition of the 3′ primer corresponding to the anti-sense strand of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 3′ primermay be desired in certain circumstances (e.g., stop codon sequences,etc.). The skilled artisan would appreciate that modifications of theabove nucleotide positions may be necessary for optimizing PCRamplification.

[1135] The same general formulas provided above may be used inidentifying the 5′ and 3′ primer sequences for amplifying any C-terminaldeletion mutant of the present invention. Moreover, the same generalformulas provided above may be used in identifying the 5′ and 3′ primersequences for amplifying any combination of N-terminal and C-terminaldeletion mutant of the present invention. The skilled artisan wouldappreciate that modifications of the above nucleotide positions may benecessary for optimizing PCR amplification.

Example 19

[1136] Regulation of Protein Expression Via Controlled Aggregation inthe Endoplasmic Reticulum

[1137] As described more particularly herein, proteins regulate diversecellular processes in higher organisms, ranging from rapid metabolicchanges to growth and differentiation. Increased production of specificproteins could be used to prevent certain diseases and/or diseasestates. Thus, the ability to modulate the expression of specificproteins in an organism would provide significant benefits.

[1138] Numerous methods have been developed to date for introducingforeign genes, either under the control of an inducible, constitutivelyactive, or endogenous promoter, into organisms. Of particular interestare the inducible promoters (see, M. Gossen, et al., Proc. Natl. Acad.Sci. USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA,91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346(1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in additionto additional examples disclosed elsewhere herein). In one example, thegene for erthropoietin (Epo) was transferred into mice and primatesunder the control of a small molecule inducer for expression (e.g.,tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X.Ye etal., Science, 283:88 (1999). Although such systems enable efficientinduction of the gene of interest in the organism upon addition of theinducing agent (i.e., tetracycline, rapamycin, etc,.), the levels ofexpression tend to peak at 24 hours and trail off to background levelsafter 4 to 14 days. Thus, controlled transient expression is virtuallyimpossible using these systems, though such control would be desirable.

[1139] A new alternative method of controlling gene expression levels ofa protein from a transgene (i.e., includes stable and transienttransformants) has recently been elucidated (V. M. Rivera., et al.,Science, 287:826-830, (2000)). This method does not control geneexpression at the level of the mRNA like the aforementioned systems.Rather, the system controls the level of protein in an active secretedform. In the absence of the inducing agent, the protein aggregates inthe ER and is not secreted. However, addition of the inducing agentresults in dis-aggregation of the protein and the subsequent secretionfrom the ER. Such a system affords low basal secretion, rapid, highlevel secretion in the presence of the inducing agent, and rapidcessation of secretion upon removal of the inducing agent. In fact,protein secretion reached a maximum level within 30 minutes ofinduction, and a rapid cessation of secretion within 1 hour of removingthe inducing agent. The method is also applicable for controlling thelevel of production for membrane proteins.

[1140] Detailed methods are presented in V. M. Rivera., et al., Science,287:826-830, (2000)), briefly:

[1141] Fusion protein constructs are created using polynucleotidesequences of the present invention with one or more copies (preferablyat least 2, 3, 4, or more) of a conditional aggregation domain (CAD) adomain that interacts with itself in a ligand-reversible manner (i.e.,in the presence of an inducing agent) using molecular biology methodsknown in the art and discussed elsewhere herein. The CAD domain may bethe mutant domain isolated from the human FKBP12 (Phe36 to Met) protein(as disclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), oralternatively other proteins having domains with similarligand-reversible, self-aggregation properties. As a principle of designthe fusion protein vector would contain a furin cleavage sequenceoperably linked between the polynucleotides of the present invention andthe CAD domains. Such a cleavage site would enable the proteolyticcleavage of the CAD domains from the polypeptide of the presentinvention subsequent to secretion from the ER and upon entry into thetrans-Golgi (J.B. Denault, et al., FEBS Lett., 379:113, (1996)).Alternatively, the skilled artisan would recognize that any proteolyticcleavage sequence could be substituted for the furin sequence providedthe substituted sequence is cleavable either endogenously (e.g., thefurin sequence) or exogenously (e.g., post secretion, post purification,post production, etc.). The preferred sequence of each feature of thefusion protein construct, from the 5′ to 3′ direction with each featurebeing operably linked to the other, would be a promoter, signalsequence, “X” number of (CAD)x domains, the furin sequence (or otherproteolytic sequence), and the coding sequence of the polypeptide of thepresent invention. The artisan would appreciate that the promotor andsignal sequence, independent from the other, could be either theendogenous promotor or signal sequence of a polypeptide of the presentinvention, or alternatively, could be a heterologous signal sequence andpromotor.

[1142] The specific methods described herein for controlling proteinsecretion levels through controlled ER aggregation are not meant to belimiting are would be generally applicable to any of the polynucleotidesand polypeptides of the present invention, including variants,homologues, orthologs, and fragments therein.

Example 20

[1143] Alteration of Protein Glycosylation Sites to EnhanceCharacteristics of Polypeptides of the Invention

[1144] Many eukaryotic cell surface and proteins arepost-translationally processed to incorporate N-linked and 0-linkedcarbohydrates (Kornfeld and Komfeld (1985) Annu. Rev. Biochem.54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838).Protein glycosylation is thought to serve a variety of functionsincluding: augmentation of protein folding, inhibition of proteinaggregation, regulation of intracellular trafficking to organelles,increasing resistance to proteolysis, modulation of proteinantigenicity, and mediation of intercellular adhesion (Fieldler andSimons (1995) Cell, 81:309-312; Helenius (1994) Mol. Biol. Of the Cell5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982)Cell, 37:417-427; Alexamnder and Elder (1984), Science, 226:1328-1330;and Flack et al., (1994), J. Biol. Chem., 269:14015-14020). In higherorganisms, the nature and extent of glycosylation can markedly affectthe circulating half-life and bio-availability of proteins by mechanismsinvolving receptor mediated uptake and clearance (Ashwell and Morrell,(1974), Adv. Enzymol., 41:99-128; Ashwell and Harford (1982), Ann. Rev.Biochem., 51:531-54). Receptor systems have been identified that arethought to play a major role in the clearance of serum proteins throughrecognition of various carbohydrate structures on the glycoproteins(Stockert (1995), Physiol. Rev., 75:591-609; Kery et al., (1992), Arch.Biochem. Biophys., 298:49-55). Thus, production strategies resulting inincomplete attachment of terminal sialic acid residues might provide ameans of shortening the bioavailability and half-life of glycoproteins.Conversely, expression strategies resulting in saturation of terminalsialic acid attachment sites might lengthen protein bioavailability andhalf-life.

[1145] In the development of recombinant glycoproteins for use aspharmaceutical products, for example, it has been speculated that thepharmacodynamics of recombinant proteins can be modulated by theaddition or deletion of glycosylation sites from a glycoproteins primarystructure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53).However, studies have reported that the deletion of N-linkedglycosylation sites often impairs intracellular transport and results inthe intracellular accumulation of glycosylation site variants (Machamerand Rose (1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992),J. Virology., 66:7136-7145; Collier et al., (1993), Biochem.,32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). Whileglycosylation site variants of proteins can be expressedintracellularly, it has proved difficult to recover useful quantitiesfrom growth conditioned cell culture medium.

[1146] Moreover, it is unclear to what extent a glycosylation site inone species will be recognized by another species glycosylationmachinery. Due to the importance of glycosylation in protein metabolism,particularly the secretion and/or expression of the protein, whether aglycosylation signal is recognized may profoundly determine a proteinsability to be expressed, either endogenously or recombinately, inanother organism (i.e., expressing a human protein in E. coli, yeast, orviral organisms; or an E. coli, yeast, or viral protein in human, etc.).Thus, it may be desirable to add, delete, or modify a glycosylationsite, and possibly add a glycosylation site of one species to a proteinof another species to improve the proteins functional, bioprocesspurification, and/or structural characteristics (e.g., a polypeptide ofthe present invention).

[1147] A number of methods may be employed to identify the location ofglycosylation sites within a protein. One preferred method is to run thetranslated protein sequence through the PROSITE computer program (SwissInstitute of Bioinformatics). Once identified, the sites could besystematically deleted, or impaired, at the level of the DNA usingmutagenesis methodology known in the art and available to the skilledartisan, Preferably using PCR-directed mutagenesis (See Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Similarly, glycosylation sites could be added, ormodified at the level of the DNA using similar methods, preferably PCRmethods (See, Maniatis, supra). The results of modifying theglycosylation sites for a particular protein (e.g., solubility,secretion potential, activity, aggregation, proteolytic resistance,etc.) could then be analyzed using methods know in the art.

Example 21

[1148] Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention through Molecular Evolution

[1149] Although many of the most biologically active proteins known arehighly effective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, and/or industrial applications. Among these traits, a shortphysiological half-life is the most prominent problem, and is presenteither at the level of the protein, or the level of the proteins mRNA.The ability to extend the half-life, for example, would be particularlyimportant for a proteins use in gene therapy, transgenic animalproduction, the bioprocess production and purification of the protein,and use of the protein as a chemical modulator among others. Therefore,there is a need to identify novel variants of isolated proteinspossessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

[1150] Thus; one aspect of the present invention relates to the abilityto enhance specific characteristics of invention through directedmolecular evolution. Such an enhancement may, in a non-limiting example,benefit the inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

[1151] For example, an engineered transient potential receptor may beconstitutively active upon binding of its cognate ligand. Alternatively,an engineered transient potential receptor may be constitutively activein the absence of ligand binding. In yet another example, an engineeredtransient potential receptor may be capable of being activated with lessthan all of the regulatory factors and/or conditions typically requiredfor transient potential receptor activation (e.g., ligand binding,phosphorylation, conformational changes, etc.). Such transient potentialreceptors would be useful in screens to identify transient potentialreceptor modulators, among other uses described herein.

[1152] Directed evolution is comprised of several steps. The first stepis to establish a library of variants for the gene or protein ofinterest. The most important step is to then select for those variantsthat entail the activity you wish to identify. The design of the screenis essential since your screen should be selective enough to eliminatenon-useful variants, but not so stringent as to eliminate all variants.The last step is then to repeat the above steps using the best variantfrom the previous screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

[1153] Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

[1154] Random mutagenesis has been the most widely recognized method todate. Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

[1155] While both of the aforementioned methods are effective forcreating randomized pools of macromolecule variants, a third method,termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747,(1994)) has recently been elucidated. DNA shuffling has also beenreferred to as “directed molecular evolution”, “exon-shuffling”,“directed enzyme evolution”, “in vitro evolution”, and “artificialevolution”. Such reference terms are known in the art and areencompassed by the invention. This new, preferred, method apparentlyovercomes the limitations of the previous methods in that it not onlypropagates positive traits, but simultaneously eliminates negativetraits in the resulting progeny.

[1156] DNA shuffling accomplishes this task by combining the principalof in vitro recombination, along with the method of “error-prone” PCR.In effect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide of interest -regions not typically accessible via hybridization of the entirepolynucleotide. Moreover, since the PCR assembly reaction utilizes“error-prone” PCR reaction conditions, random mutations are introducedduring the DNA synthesis step of the PCR reaction for all of thefragments -further diversifying the potential hybridization sites duringthe annealing step of the reaction.

[1157] A variety of reaction conditions could be utilized to carry-outthe DNA shuffling reaction. However, specific reaction conditions forDNA shuffling are provided, for example, in PNAS, 91:10747, (1994).Briefly:

[1158] Prepare the DNA substrate to be subjected to the DNA shufflingreaction. Preparation may be in the form of simply purifying the DNAfrom contaminating cellular material, chemicals, buffers,oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entailthe use of DNA purification kits as those provided by Qiagen, Inc., orby the Promega, Corp., for example.

[1159] Once the DNA substrate has been purified, it would be subjectedto Dnase I digestion. About 2-4 ug of the DNA substrate(s) would bedigested with .0015 units of Dnase I (Sigma) per ul in 100 ul of 5OmMTris-HCL, pH 7.4/l mM MgC12 for 10-20 min. at room temperature. Theresulting fragments of 10-50bp could then be purified by running themthrough a 2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50bp fragments could be eluted from said paper using IM NaCl,followed by ethanol precipitation.

[1160] The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, lOmM Tris.HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60s; 94 C for 30s, 50-55 C for 30s, and 72 C for 30s using 30-45cycles, followed by 72 C for Smin using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primeness product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30s, 50 C for 30s, and72 C for 30s). The referred primers would be primers corresponding tothe nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

[1161] The resulting shuffled, assembled, and amplified product can bepurified using methods well known in the art (e.g., Qiagen PCRpurification kits) and then subsequently cloned using appropriaterestriction enzymes.

[1162] Although a number of variations of DNA shuffling have beenpublished to date, such variations would be obvious to the skilledartisan and are encompassed by the invention. The DNA shuffling methodcan also be tailored to the desired level of mutagenesis using themethods described by Zhao, et al. (Nucl Acid Res., 25(6): 1307-1308,(1997).

[1163] As described above, once the randomized pool has been created, itcan then be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

[1164] DNA shuffling has several advantages. First, it makes use ofbeneficial mutations. When combined with screening, DNA shuffling allowsthe discovery of the best mutational combinations and does not assumethat the best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

[1165] A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

[1166] Finally, recombination enables parallel processing. Thisrepresents a significant advantage since there are likely multiplecharacteristics that would make a protein more desirable (e.g.solubility, activity, etc.). Since it is increasingly difficult toscreen for more than one desirable trait at a time, other methods ofmolecular evolution tend to be inhibitory. However, using recombination,it would be possible to combine the randomized fragments of the bestrepresentative variants for the various traits, and then select formultiple properties at once.

[1167] DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self′ molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

[1168] Likewise, the invention encompasses the application of DNAshuffling technology to the evolution of polynucleotides andpolypeptides of the invention, wherein one or more cycles of DNAshuffling include, in addition to the gene template DNA,oligonucleotides coding for known allelic sequences, optimized codonsequences, known variant sequences, known polynucleotide polymorphismsequences, known ortholog sequences, known homologue sequences,additional homologous sequences, additional non-homologous sequences,sequences from another species, and any number and combination of theabove.

[1169] In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

[1170] Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 22

[1171] Method of Determining Alterations in a Gene Corresponding to aPolynucleotide

[1172] RNA isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease) is beisolated. cDNA is then generated from these RNA samples using protocolsknown in the art. (See, Sambrook.) The cDNA is then used as a templatefor PCR, employing primers surrounding regions of interest in SEQ IDNO:1. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70degrees C., using buffer solutions described in Sidransky et al.,Science 252:706 (1991).

[1173] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

[1174] PCR products are cloned into T-tailed vectors as described inHolton et al., Nucleic Acids Research, 19: 156 (1991) and sequenced withT7 polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

[1175] Genomic rearrangements are also observed as a method ofdetermining alterations in a gene corresponding to a polynucleotide.Genomic clones isolated according to Example 11 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

[1176] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, NC.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 23

[1177] Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

[1178] A polypeptide of the present invention can be detected in abiological sample, and if an increased or decreased level of thepolypeptide is detected, this polypeptide is a marker for a particularphenotype. Methods of detection are numerous, and thus, it is understoodthat one skilled in the art can modify the following assay to fit theirparticular needs.

[1179] For example, antibody-sandwich ELISAs are used to detectpolypeptides in a sample, preferably a biological sample. Wells of amicrotiter plate are coated with specific antibodies, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced by the method described elsewhere herein.The wells are blocked so that non-specific binding of the polypeptide tothe well is reduced.

[1180] The coated wells are then incubated for >2 hours at RT with asample containing the polypeptide. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedpolypeptide.

[1181] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[1182] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by amicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot polypeptide concentration on theX-axis (log scale) and fluorescence or absorbance of the Y-axis (linearscale). Interpolate the concentration of the polypeptide in the sampleusing the standard curve.

Example 24

[1183] Formulation

[1184] The invention also provides methods of treatment and/orprevention diseases, disorders, and/or conditions (such as, for example,any one or more of the diseases or disorders disclosed herein) byadministration to a subject of an effective amount of a Therapeutic. Bytherapeutic is meant a polynucleotides or polypeptides of the invention(including fragments and variants), agonists or antagonists thereof,and/or antibodies thereto, in combination with a pharmaceuticallyacceptable carrier type (e.g., a sterile carrier).

[1185] The Therapeutic will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the Therapeutic alone), the site of delivery, the methodof administration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

[1186] As a general proposition, the total pharmaceutically effectiveamount of the Therapeutic administered parenterally per dose will be inthe range of about lug/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Therapeutic is typicallyadministered at a dose rate of about I ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[1187] Therapeutics can be administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[1188] In yet an additional embodiment, the Therapeutics of theinvention are delivered orally using the drug delivery technologydescribed in U.S. Pat. No. 6,258,789, which is hereby incorporated byreference herein.

[1189] In yet an additional embodiment, the Therapeutics of theinvention are delivered orally using the drug delivery technologydescribed in U.S. Pat. No. 6,258,789, which is hereby incorporated byreference herein.

[1190] Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[1191] Therapeutics of the invention may also be suitably administeredby sustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

[1192] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2- hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

[1193] Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see, generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317 -327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. AppI. 83-I18008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

[1194] In yet an additional embodiment, the Therapeutics of theinvention are delivered by way of a pump (see Langer, supra; Sefton, CRCCrit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[1195] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[1196] For parenteral administration, in one embodiment, the Therapeuticis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

[1197] Generally, the formulations are prepared by contacting theTherapeutic uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[1198] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[1199] The Therapeutic will typically be formulated in such vehicles ata concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml,at a pH of about 3 to 8. It will be understood that the use of certainof the foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[1200] Any pharmaceutical used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

[1201] Therapeutics ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

[1202] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the Therapeutics of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, theTherapeutics may be employed in conjunction with other therapeuticcompounds.

[1203] The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax lOOa, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[1204] The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[1205] In one embodiment, the Therapeutics of the invention areadministered in combination with members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with theTherapeutics of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892),TR1O (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

[1206] In certain embodiments, Therapeutics of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR(zidovudine/AZT), VIDEX (didanosine/ddI), HIVID (zalcitabine/ddC), ZERIT(stavudine/d4T), EPIVIR (lamivudine/3TC), and COMBIVIR(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE (nevirapine),RESCRIPTOR (delavirdine), and SUSTIVA (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN (indinavir), NORVIR(ritonavir), INVIRASE (saquinavir), and VIRACEPT (nelfinavir). In aspecific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HW infection.

[1207] In other embodiments, Therapeutics of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, ATOVAQUONE,ISONIAZID, RIFAMPIN, PYRAZINAMIDE, ETHAMBUTOL, RIFABUTIN,CLARITHROMYCIN, AZITHROMYCIN, GANCICLOVIR, FOSCARNET, CIDOFOVIR,FLUCONAZOLE, ITRACONAZOLE, KETOCONAZOLE, ACYCLOVIR, FAMCICOLVIR,PYRIMETHAMINE, LEUCOVORIN, NEUPOGEN (filgrastim/G-CSF), and LEUKINE(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, and/or ATOVAQUONEto prophylactically treat or prevent an opportunistic Pneumocystiscarinii pneumonia infection. In another specific embodiment,Therapeutics of the invention are used in any combination withISONIAZID, RIFAMPIN, PYRAZINAMIDE, and/or ETHAMBUTOL to prophylacticallytreat or prevent an opportunistic Mycobacterium avium complex infection.In another specific embodiment, Therapeutics of the invention are usedin any combination with RIFABUTIN, CLARITHROMYCIN, and/or AZITHROMYCINto prophylactically treat or prevent an opportunistic Mycobacteriumtuberculosis infection. In another specific embodiment, Therapeutics ofthe invention are used in any combination with GANCICLOVIR, FOSCARNET,and/or CIDOFOVIR to prophylactically treat or prevent an opportunisticcytomegalovirus infection. In another specific embodiment, Therapeuticsof the invention are used in any combination with FLUCONAZOLE,ITRACONAZOLE, and/or KETOCONAZOLE to prophylactically treat or preventan opportunistic fungal infection. In another specific embodiment,Therapeutics of the invention are used in any combination with ACYCLOVIRand/or FAMCICOLVIR to prophylactically treat or prevent an opportunisticherpes simplex virus type I and/or type II infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with PYRIMETHAMINE and/or LEUCOVORIN to prophylacticallytreat or prevent an opportunistic Toxoplasma gondii infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with LEUCOVORIN and/or NEUPOGEN to prophylacticallytreat or prevent an opportunistic bacterial infection.

[1208] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[1209] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

[1210] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[1211] In specific embodiments, Therapeutics of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the Therapeutics of theinvention include, but are not limited to, ORTHOCLONE (OKT3),SANDIMMUNE/NEORAL/SANGDYA (cyclosporin), PROGRAF (tacrolimus), CELLCEPT(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE(sirolimus). In a specific embodiment, immunosuppressants may be used toprevent rejection of organ or bone marrow transplantation.

[1212] In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR, IVEEGAM, SANDOGLOBULIN, GAMMAGARD S/D, and GAMIMUNE.In a specific embodiment, Therapeutics of the invention are administeredin combination with intravenous immune globulin preparations intransplantation therapy (e.g., bone marrow transplant).

[1213] In an additional embodiment, the Therapeutics of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with theTherapeutics of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[1214] In another embodiment, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[1215] In a specific embodiment, Therapeutics of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or any combination of the components ofCHOP. In another embodiment, Therapeutics of the invention areadministered in combination with Rituximab. In a further embodiment,Therapeutics of the invention are administered with Rituxmab and CHOP,or Rituxmab and any combination of the components of CHOP.

[1216] In an additional embodiment, the Therapeutics of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, I12, IL13, IL 5,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-lalpha, IL-lbeta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-l9, IL-20, and IL-21.

[1217] In an additional embodiment, the Therapeutics of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

[1218] In an additional embodiment, the Therapeutics of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE(SARGRAMOSTIM) and NEUPOGEN (FILGRASTIM).

[1219] In an additional embodiment, the Therapeutics of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the Therapeuticsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[1220] In an additional embodiment, the Therapeutics of the inventionare administered in combination with other immune factors. Immunefactors that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Ly9, CD2, CD48, CD58, 2B4, CD84,CDw15O, CTLA4, CTLA4Ig, Bsl1, Bsl2, Bs13, BLYS, TRAIL, APRIL, B7, B7antagonists, B7 agonists, and Ret16.

[1221] In a specific embodiment, formulations of the present inventionmay further comprise antagonists of P-glycoprotein (also referred to asthe multiresistance protein, or PGP), including antagonists of itsencoding polynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). P-glycoprotein is well known for decreasingthe efficacy of various drug administrations due to its ability toexport intracellular levels of absorbed drug to the cell exterior. Whilethis activity has been particularly pronounced in cancer cells inresponse to the administration of chemotherapy regimens, a variety ofother cell types and the administration of other drug classes have beennoted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations inthe PGP gene significantly reduces PGP function, making it less able toforce drugs out of cells. People who have two versions of the mutatedgene—one inherited from each parent—have more than four times less PGPthan those with two normal versions of the gene. People may also haveone normal gene and one mutated one. Certain ethnic populations haveincreased incidence of such PGP mutations. Among individuals from Ghana,Kenya, the Sudan, as well as African Americans, frequency of the normalgene ranged from 73% to 84%. In contrast, the frequency was 34% to 59%among British whites, Portuguese, Southwest Asian, Chinese, Filipino andSaudi populations. As a result, certain ethnic populations may requireincreased administration of PGP antagonist in the formulation of thepresent invention to arrive at the an efficacious dose of thetherapeutic (e.g., those from African descent). Conversely, certainethnic populations, particularly those having increased frequency of themutated PGP (e.g., of Caucasian descent, or non-African descent) mayrequire less pharmaceutical compositions in the formulation due to aneffective increase in efficacy of such compositions as a result of theincreased effective absorption (e.g., less PGP activity) of saidcomposition.

[1222] Moreover, in another specific embodiment, formulations of thepresent invention may further comprise antagonists of OATP2 (alsoreferred to as the multiresistance protein, or MRP2), includingantagonists of its encoding polynucleotides (e.g., antisenseoligonucleotides, ribozymes, zinc-finger proteins, etc.). The inventionalso further comprises any additional antagonists known to inhibitproteins thought to be attributable to a multidrug resistant phenotypein proliferating cells.

[1223] Preferred antagonists that formulations of the present maycomprise include the potent P-glycoprotein inhibitor elacridar, and/orLY-335979. Other P-glycoprotein inhibitors known in the art are alsoencompassed by the present invention.

[1224] In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 25

[1225] Method of Treating Decreased Levels of the Polypeptide

[1226] The present invention relates to a method for treating anindividual in need of an increased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an agonistof the invention (including polypeptides of the invention). Moreover, itwill be appreciated that conditions caused by a decrease in the standardor normal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

[1227] For example, a patient with decreased levels of a polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided herein.

Example 26

[1228] Method of Treating Increased Levels of the Polypeptide

[1229] The present invention also relates to a method of treating anindividual in need of a decreased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of anantagonist of the invention (including polypeptides and antibodies ofthe invention).

[1230] In one example, antisense technology is used to inhibitproduction of a polypeptide of the present invention. This technology isone example of a method of decreasing levels of a polypeptide,preferably a secreted form, due to a variety of etiologies, such ascancer. For example, a patient diagnosed with abnormally increasedlevels of a polypeptide is administered intravenously antisensepolynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days.This treatment is repeated after a 7-day rest period if the treatmentwas well tolerated. The formulation of the antisense polynucleotide isprovided herein.

Example 27

[1231] Method of Treatment Using Gene Therapy-Ex Vivo

[1232] One method of gene therapy transplants fibroblasts, which arecapable of expressing a polypeptide, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37 degreeC. for approximately one week.

[1233] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[1234] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[1235] The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 11 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

[1236] The amphotropic pA317 or GP+aml2 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[1237] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether protein is produced.

[1238] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 28

[1239] Gene Therapy Using Endogenous Genes Corresponding ToPolynucleotides of the Invention

[1240] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

[1241] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

[1242] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[1243] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[1244] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous polynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

[1245] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM +10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

[1246] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the locus correspondingto the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas,Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplifiedby PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end.Two non-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindIII site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHl; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

[1247] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×106cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960pF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[1248] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

[1249] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 29

[1250] Method of Treatment Using Gene Therapy—In Vivo

[1251] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences into an animal to increase ordecrease the expression of the polypeptide. The polynucleotide of thepresent invention may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the polypeptide by thetarget tissue. Such gene therapy and delivery techniques and methods areknown in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat.Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

[1252] The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically,acceptable liquid or aqueous carrier.

[1253] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

[1254] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[1255] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[1256] For the naked polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[1257] The dose response effects of injected polynucleotide in muscle invivo is determined as follows. Suitable template DNA for production ofmRNA coding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[1258] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The template DNA is injected in 0.1 ml of carrierin a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[1259] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 30

[1260] Transgenic Animals

[1261] The polypeptides of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[1262] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of thepolynucleotides of the invention using a gene gun (see, e.g., Ulmer etal., Science 259:1745 (1993); introducing nucleic acid constructs intoembryonic pleuripotent stem cells and transferring the stem cells backinto the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[1263] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[1264] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[1265] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[1266] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[1267] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of polypeptides of the present invention, studyingdiseases, disorders, and/or conditions associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch diseases, disorders, and/or conditions.

Example 31

[1268] Knock-Out Animals

[1269] Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

[1270] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

[1271] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[1272] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[1273] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 32

[1274] Method of Isolating Antibody Fragments Directed Against TRP-PLIK2polypeptides, TRP-PLIK2b, TRP-PLIK2c, and/or TRP-PLIK2d From A LibraryOf scFvs

[1275] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against TRP-PLIK2 polypeptides, TRP-PLIK2b, TRP-PLIK2c,and/or TRP-PLIK2d to which the donor may or may not have been exposed(see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference inits entirety).

[1276] Rescue of the Library. A library of scFvs is constructed from theRNA of human PBLs as described in PCT publication WO 92/01047. To rescuephage displaying antibody fragments, approximately 109 E. coli harboringthe phagemid are used to inoculate 50 ml of 2×TY containing 1% glucoseand 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8with shaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2 ×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

[1277] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[1278] Panning of the Library. Immunotubes (Nunc) are coated overnightin PBS with 4 ml of either 100 μg/ml or 10 g/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100pg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[1279] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scFvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

[1280] Moreover, in another preferred method, the antibodies directedagainst the polypeptides of the present invention may be produced inplants. Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

Example 33

[1281] Identification and Cloning of VH and VL domains Of AntibodiesDirected Against the TRP-PLIK2, TRP-PLIK2b, TRP-PLIK2c, and/orTRP-PLIK2d Polypeptide

[1282] VH and VL domains may be identified and cloned from cell linesexpressing an antibody directed against a TRP-PLIK2, TRP-PLIK2b,TRP-PLIK2c, and/or TRP-PLIK2d epitope by performing PCR with VH and VLspecific primers on cDNA made from the antibody expressing cell lines.Briefly, RNA is isolated from the cell lines and used as a template forRT-PCR designed to amplify the VH and VL domains of the antibodiesexpressed by the EBV cell lines. Cells may be lysed using the TRIzolreagent (Life Technologies, Rockville, Md.) and extracted with one fifthvolume of -chloroform. After addition of chloroform, the solution isallowed to incubate at room temperature for 10 minutes, and thencentrifuged at 14, 000 rpm for 15 minutes at 4 C in a tabletopcentrifuge. The supernatant is collected and RNA is precipitated usingan equal volume of isopropanol. Precipitated RNA is pelleted bycentrifuging at 14, 000 rpm for 15 minutes at 4 C in a tabletopcentrifuge.

[1283] Following centrifugation, the supernatant is discarded and washedwith 75% ethanol. Follwing the wash step, the RNA is centrifuged againat 800 rpm for 5 minutes at 4 C. The supernatant is discarded and thepellet allowed to air dry. RNA is the dissolved in DEPC water and heatedto 60 C for 10 minutes. Quantities of RNA can be determined usingoptical density measurements. cDNA may be synthesized, according tomethods well-known in the art and/or described herein, from 1. 5-2. 5micrograms of RNA using reverse transciptase and random hexamer primers.cDNA is then used as a template for PCR amplification of VH and VLdomains.

[1284] Primers used to amplify VH and VL genes are shown below.Typically a PCR reaction makes use of a single 5′primer and a single3′primer. Sometimes, when the amount of available RNA template islimiting, or for greater efficiency, groups of 5′ and/or 3′primers maybe used. For example, sometimes all five VH-5′primers and allJH3′primers are used in a single PCR reaction. The PCR reaction iscarried out in a 50 microliter volume containing 1×PCR buffer, 2 mM ofeach dNTP, 0. 7 units of High Fidelity Taq polymerse, 5′primer mix,3′primer mix and 7. 5 microliters of cDNA. The 5′ and 3′primer mix ofboth VH and VL can be made by pooling together 22 pmole and 28 pmole,respectively, of each of the individual primers. PCR conditions are: 96C for 5 minutes ; followed by 25 cycles of 94 C for 1 minute, 50 C for 1minute, and 72 C for 1 minute ; followed by an extension cycle of 72 Cfor 10 minutes. After the reaction has been completed, sample tubes maybe stored at 4 C. SEQ ID Primer name Primer Sequence NO: PrimerSequences Used to Amplify VH domains. Hu VH1 -5′ CAGGTGCAGCTGGTGCAGTCTGG272 Hu VH2 -5′ CAGGTCAACTTAAGGGAGTCTGG 273 Hu VH3 -5′GAGGTGCAGCTGGTGGAGTCTGG 274 Hu VH4 -5′ CAGGTGCAGCTGCAGGAGTCGGG 275 HuVHS -5′ GAGGTGCAGCTGTTGCAGTCTGC 276 Hu VH6 -5′ CAGGTACAGCTGCAGCAGTCAGG277 Hu JHI -5′ TGAGGAGACGGTGACCAGGGTGCC 278 Hu JH3 -5′TGAAGAGACGGTGACCATTGTCCC 279 Hu JH4 -5′ TGAGGAGACGGTGACCAGGGTTCC 280 HuJH6 -5′ TGAGGAGACGGTGACCGTGGTCCC 281 Primer Sequences Used to Amplify VLdomains Hu Vkappa1 -5′ GACATCCAGATGACCCAGTCTCC 282 Hu Vkappa2a -5′GATGTTGTGATGACTCAGTCTCC 283 Hu Vkappa2b -5′ GATATTGTGATGACTCAGTCTCC 284Hu Vkappa3 -5′ GAAATTGTGTTGACGCAGTCTCC 285 Hu Vkappa4 -5′GACATCGTGATGACCCAGTCTCC 286 Hu Vkappa5 -5′ GAAACGACACTCACGCAGTCTCC 287Hu Vkappa6 -5′ GAAATTGTGCTGACTCAGTCTCC 288 Hu Vlambda1 -5′CAGTCTGTGTTGACGCAGCCGCC 289 Hu Vlambda2 -5′ CAGTCTGCCCTGACTCAGCCTGC 290Hu Vlambda3 -5′ TCCTATGTGCTGACTCAGCCACC 291 Hu Vlambda3b -5′TCTTCTGAGCTGACTCAGGACCC 292 Hu Vlambda4 -5′ CACGTTATACTGACTCAACCGCC 293Hu Vlambda5 -5′ CAGGCTGTGCTCACTCAGCCGTC 294 Hu Vlambda6 -5′AATTTTATGCTGACTCAGCCCCA 295 Hu Jkappa1 -3′ ACGTTTGATTTCCACCTTGGTCCC 296Hu Jkappa2 -3′ ACGTTTGATCTCCAGCTTGGTCCC 297 Hu Jkappa3 -3′ACGTTTGATATCCACTTTGGTCCC 298 Hu Jkappa4 -3′ ACGTTTGATCTCCACCTTGGTCCC 299Hu Jkappa5 -3′ ACGTTTAATCTCCAGTCGTGTCCC 300 Hu Vlambda1 -3′CAGTCTGTGTTGACGCAGCCGCC 301 Hu Vlambda2 -3′ CAGTCTGCCCTGACTCAGCCTGC 302Hu Vlambda3 -3′ TCCTATGTGCTGACTCAGCCACC 303 Hu Vlambda3b -3′TCTTCTGAGCTGACTCAGGACCC 304 Hu Vlambda4 -3′ CACGTTATACTGACTCAACCGCC 305Hu Vlambda5 -3′ CAGGCTGTGCTCACTCAGCCGTC 306 Hu Vlambda6 -3′AATTTTATGCTGACTCAGCCCCA 307

[1285] PCR samples are then electrophoresed on a 1. 3% agarose gel. DNAbands of the expected sizes (-506 base pairs for VH domains, and 344base pairs for VL domains) can be cut out of the gel and purified usingmethods well known in the art and/or described herein.

[1286] Purified PCR products can be ligated into a PCR cloning vector(TA vector from Invitrogen Inc., Carlsbad, Calif.). Individual clonedPCR products can be isolated after transfection of E. coli andblue/white color selection. Cloned PCR products may then be sequencedusing methods commonly known in the art and/or described herein.

[1287] The PCR bands containing the VH domain and the VL domains canalso be used to create full-length Ig expression vectors. VH and VLdomains can be cloned into vectors containing the nucleotide sequencesof a heavy (e. g., human IgGI or human IgG4) or light chain (human kappaor human ambda) constant regions such that a complete heavy or lightchain molecule could be expressed from these vectors when transfectedinto an appropriate host cell. Further, when cloned heavy and lightchains are both expressed in one cell line (from either one or twovectors), they can assemble into a complete functional antibody moleculethat is secreted into the cell culture medium. Methods usingpolynucleotides encoding VH and VL antibody domain to generateexpression vectors that encode complete antibody molecules are wellknown within the art.

Example 34

[1288] Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

[1289] Generation of functional humoral immune responses requires bothsoluble and cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

[1290] One of the best studied classes of B-cell co-stimulatory proteinsis the TNF-superfamily. Within this family CD40, CD27, and CD30 alongwith their respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

[1291] In Vitro Assay- Purified polypeptides of the invention, ortruncated forms thereof, is assessed for its ability to induceactivation, proliferation, differentiation or inhibition and/or death inB-cell populations and their precursors. The activity of thepolypeptides of the invention on purified human tonsillar B cells,measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, isassessed in a standard B-lymphocyte co-stimulation assay in whichpurified tonsillar B cells are cultured in the presence of eitherformalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilizedanti-human IgM antibody as the priming agent. Second signals such asIL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cellproliferation as measured by tritiated-thymidine incorporation. Novelsynergizing agents can be readily identified using this assay. The assayinvolves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).

[1292] Various dilutions of each sample are placed into individual wellsof a 96-well plate to which are added 105 B-cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10-5M 2ME, 100 U/ml penicillin,10 ug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of150 ul. Proliferation or inhibition is quantitated by a 20h pulse(luCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

[1293] In Vivo Assay- BALB/c mice are injected (i.p.) twice per day withbuffer only, or 2 mg/Kg of a polypeptide of the invention, or truncatedforms thereof. Mice receive this treatment for 4 consecutive days, atwhich time they are sacrificed and various tissues and serum collectedfor analyses. Comparison of H&E sections from normal spleens and spleenstreated with polypeptides of the invention identify the results of theactivity of the polypeptides on spleen cells, such as the diffusion ofperi-arterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

[1294] Flow cytometric analyses of the spleens from mice treated withpolypeptide is used to indicate whether the polypeptide specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

[1295] Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andpolypeptide-treated mice.

[1296] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 35

[1297] T Cell Proliferation Assay

[1298] A CD3-induced proliferation assay is performed on PBMCs and ismeasured by the uptake of 3H-thymidine. The assay is performed asfollows. Ninety-six well plates are coated with 100 (1well of mAb to CD3(HIT3a, Pharmingen) or isotype-matched control mAb (B33. 1) overnight at4 degrees C. (I (g/ml in .05M bicarbonate buffer, pH 9.5), then washedthree times with PBS. PBMC are isolated by F/H gradient centrifugationfrom human peripheral blood and added to quadruplicate wells (5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S inthe presence of varying concentrations of polypeptides of the invention(total volume 200 ul). Relevant protein buffer and medium alone arecontrols. After 48 hr. culture at 37 degrees C., plates are spun for 2min. at 1000 rpm and 100 (1 of supernatant is removed and stored −20degrees C. for measurement of IL-2 (or other cytokines) if effect onproliferation is observed. Wells are supplemented with 100 ul of mediumcontaining 0.5 uCi of 3H-thymidine and cultured at 37 degrees C. for18-24 hr. Wells are harvested and incorporation of 3H-thymidine used asa measure of proliferation. Anti-CD3 alone is the positive control forproliferation. IL-2 (100 U/ml) is also used as a control which enhancesproliferation. Control antibody which does not induce proliferation of Tcells is used as the negative controls for the effects of polypeptidesof the invention.

[1299] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 36

[1300] Effect of Polypeptides of the Invention on the Expression of MHCClass II, Costimulatory and Adhesion Molecules and Cell Differentiationof Monocytes and Monocyte-Derived Human Dendritic Cells

[1301] Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFC(RII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

[1302] FACS analysis of surface antigens is performed as follows. Cellsare treated 1-3 days with increasing concentrations of polypeptides ofthe invention or LPS (positive control), washed with PBS containing 1%BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at4 degrees C.. After an additional wash, the labeled cells are analyzedby flow cytometry on a FACScan (Becton Dickinson).

[1303] Effect on the production of cytokines. Cytokines generated bydendritic cells, in particular IL-12, are important in the initiation ofT-cell dependent immune responses. IL-12 strongly influences thedevelopment of Thl helper T-cell immune response, and induces cytotoxicT and NK cell function. An ELISA is used to measure the IL-12 release asfollows. Dendritic cells (106/ml) are treated with increasingconcentrations of polypeptides of the invention for 24 hours. LPS (100ng/ml) is added to the cell culture as positive control. Supernatantsfrom the cell cultures are then collected and analyzed for IL-12 contentusing commercial ELISA kit(e.g., R & D Systems (Minneapolis, MN)). Thestandard protocols provided with the kits are used.

[1304] Effect on the expression of MHC Class II, costimulatory andadhesion molecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fe receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fe receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

[1305] FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofpolypeptides of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C.. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

[1306] Monocyte activation and/or increased survival. Assays formolecules that activate (or alternatively, inactivate) monocytes and/orincrease monocyte survival (or alternatively, decrease monocytesurvival) are known in the art and may routinely be applied to determinewhether a molecule of the invention functions as an inhibitor oractivator of monocytes. Polypeptides, agonists, or antagonists of theinvention can be screened using the three assays described below. Foreach of these assays, Peripheral blood mononuclear cells (PBMC) arepurified from single donor leukopacks (American Red Cross, Baltimore,Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytesare isolated from PBMC by counterflow centrifugal elutriation.

[1307] Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from internally regulated process(apoptosis). Addition to the culture of activating factors, such asTNF-alpha dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of the compound to be tested. Cells aresuspended at a concentration of 2 ×106/ml in PBS containing PI at afinal concentration of 5 (g/ml, and then incubated at room temperaturefor 5 minutes before FACScan analysis. PI uptake has been demonstratedto correlate with DNA fragmentation in this experimental paradigm.

[1308] Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5x105 cells/mlwith increasing concentrations of the a polypeptide of the invention andunder the same conditions, but in the absence of the polypeptide. ForIL-12 production, the cells are primed overnight with IFN (100 U/mI) inpresence of a polypeptide of the invention. LPS (10 ng/ml) is thenadded. Conditioned media are collected after 24h and kept frozen untiluse. Measurement of TNF-alpha, IL-10, MCP-I and IL-8 is then performedusing a commercially available ELISA kit(e.g., R & D Systems(Minneapolis, Mn.)) and applying the standard protocols provided withthe kit.

[1309] Oxidative burst. Purified monocytes are plated in 96-w plate at2-ixiO5 cell/well. Increasing concentrations of polypeptides of theinvention are added to the wells in a total volume of 0.2 ml culturemedium (RPMI 1640 +10% FCS, glutamine and antibiotics). After 3 daysincubation, the plates are centrifuged and the medium is removed fromthe wells. To the macrophage monolayers, 0.2 ml per well of phenol redsolution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mMdextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, togetherwith the stimulant (200 nM PMass.). The plates are incubated at 37(C for2 hours and the reaction is stopped by adding 20 p1 IN NaOH per well.The absorbance is read at 610 nm. To calculate the amount of H202produced by the macrophages, a standard curve of a H202 solution ofknown molarity is performed for each experiment.

[1310] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 37

[1311] Suppression of TNF Alpha-Induced Adhesion Molecule Expression bya Polypeptide of the Invention

[1312] The recruitment of lymphocytes to areas of inflammation andangiogenesis involves specific receptor-ligand interactions between cellsurface adhesion molecules (CAMs) on lymphocytes and the vascularendothelium. The adhesion process, in both normal and pathologicalsettings, follows a multi-step cascade that involves intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and endothelial leukocyte adhesion molecule-I (E-selectin)expression on endothelial cells (EC). The expression of these moleculesand others on the vascular endothelium determines the efficiency withwhich leukocytes may adhere to the local vasculature and extravasateinto the local tissue during the development of an inflammatoryresponse. The local concentration of cytokines and growth factorparticipate in the modulation of the expression of these CAMs.

[1313] Tumor necrosis factor alpha (TNF-a), a potent proinflammatorycytokine, is a stimulator of all three CAMs on endothelial cells and maybe involved in a wide variety of inflammatory responses, often resultingin a pathological outcome.

[1314] The potential of a polypeptide of the invention to mediate asuppression of TNF-a induced CAM expression can be examined. A modifiedELISA assay which uses ECs as a solid phase absorbent is employed tomeasure the amount of CAM expression on TNF-a treated ECs whenco-stimulated with a member of the FGF family of proteins.

[1315] To perform the experiment, human umbilical vein endothelial cell(HUVEC) cultures are obtained from pooled cord harvests and maintainedin growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidifiedincubator containing 5% C02. HUVECs are seeded in 96-well plates atconcentrations of 1×104 cells/well in EGM medium at 37 degree C. for18-24 hrs or until confluent. The monolayers are subsequently washed 3times with a serum-free solution of RPM1- 1640 supplemented with 100U/ml penicillin and 100 mg/ml streptomycin, and treated with a givencytokine and/or growth factor(s) for 24 h at 37 degree C. Followingincubation, the cells are then evaluated for CAM expression.

[1316] Human Umbilical Vein Endothelial cells (HUVECs) are grown in astandard 96 well plate to confluence. Growth medium is removed from thecells and replaced with 90 ul of 199 Medium (10% FBS). Samples fortesting and positive or negative controls are added to the plate intriplicate (in 10 ul volumes). Plates are incubated at 37 degree C. foreither 5 h (selectin and integrin expression) or 24 h (integrinexpression only). Plates are aspirated to remove medium and 100 μl of0.1% paraformaldehyde-PBS(with Ca++ and Mg++ ) is added to each well.Plates are held at 4oC for 30 min.

[1317] Fixative is then removed from the wells and wells are washed1×with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry.Add 10 μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 pg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37oC for 30 min. in a humidifiedenvironment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.

[1318] Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphatase(1:5,000 dilution) to each well and incubated at 37oC for 30 min. Wellsare washed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-NitrophenolPhosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μlof pNPP substrate in glycine buffer is added to each test well. Standardwells in triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphatase in glycine buffer: 1:5,000(100) >10-0.5>10-1>10-1.5. 5 μl of each dilution is added to triplicatewells and the resulting AP content in each well is 5.50 ng, 1.74 ng,0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each ofthe standard wells. The plate must be incubated at 37oC for 4h. A volumeof 50 μl of 3M NaOH is added to all wells. The results are quantified ona plate reader at 405 nm. The background subtraction option is used onblank wells filled with glycine buffer only. The template is set up toindicate the concentration of AP-conjugate in each standard well [ 5.50ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of boundAP-conjugate in each sample.

[1319] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

[1320] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[1321] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Further, the hard copy of the sequence listing submittedherewith and the corresponding computer readable form are bothincorporated herein by reference in their entireties.

1 335 1 6054 DNA Homo sapiens CDS (1)..(6051) 1 atg att atc cta tct aagtcc cag aaa tcc tgg att aaa gga gta ttt 48 Met Ile Ile Leu Ser Lys SerGln Lys Ser Trp Ile Lys Gly Val Phe 1 5 10 15 gac aag aga gaa tgt agcaca atc ata ccc agc tca aaa aat cct cac 96 Asp Lys Arg Glu Cys Ser ThrIle Ile Pro Ser Ser Lys Asn Pro His 20 25 30 aga tgt act cca gta tgc caagtc tgc cag aat tta atc agg tgt tac 144 Arg Cys Thr Pro Val Cys Gln ValCys Gln Asn Leu Ile Arg Cys Tyr 35 40 45 tgt ggc cga ctg att gga gac catgct ggg ata gat tat tcc tgg acc 192 Cys Gly Arg Leu Ile Gly Asp His AlaGly Ile Asp Tyr Ser Trp Thr 50 55 60 atc tca gct gcc aag ggt aaa gaa agtgaa caa tgg tct gtt gaa aag 240 Ile Ser Ala Ala Lys Gly Lys Glu Ser GluGln Trp Ser Val Glu Lys 65 70 75 80 cac aca acg aaa agc cca aca gat actttt ggc acg att aat ttc caa 288 His Thr Thr Lys Ser Pro Thr Asp Thr PheGly Thr Ile Asn Phe Gln 85 90 95 gat gga gag cac acc cat cat gcc aag tatatt aga act tct tat gat 336 Asp Gly Glu His Thr His His Ala Lys Tyr IleArg Thr Ser Tyr Asp 100 105 110 aca aaa ctg gat cat ctg tta cat tta atgttg aaa gag tgg aaa atg 384 Thr Lys Leu Asp His Leu Leu His Leu Met LeuLys Glu Trp Lys Met 115 120 125 gaa ctg ccc aag ctt gtg atc tca gtc catggg ggc atc cag aac ttt 432 Glu Leu Pro Lys Leu Val Ile Ser Val His GlyGly Ile Gln Asn Phe 130 135 140 act atg ccc tct aaa ttt aaa gag att ttcagc caa ggt ttg gtt aaa 480 Thr Met Pro Ser Lys Phe Lys Glu Ile Phe SerGln Gly Leu Val Lys 145 150 155 160 gct gca gag aca aca gga gcg tgg ataata act gaa ggc atc aat aca 528 Ala Ala Glu Thr Thr Gly Ala Trp Ile IleThr Glu Gly Ile Asn Thr 165 170 175 gga gtg tcc aag cat gtt ggg gat gccttg aaa tcc cat tcc tct cat 576 Gly Val Ser Lys His Val Gly Asp Ala LeuLys Ser His Ser Ser His 180 185 190 tcc ttg aga aaa atc tgg aca gtt ggaatc cct cct tgg ggt gtc att 624 Ser Leu Arg Lys Ile Trp Thr Val Gly IlePro Pro Trp Gly Val Ile 195 200 205 gag aac cag aga gac ctt att gga aaagat gtg gtg tgc ctg tac cag 672 Glu Asn Gln Arg Asp Leu Ile Gly Lys AspVal Val Cys Leu Tyr Gln 210 215 220 act ctg gat aac ccc ctc agc aag ctcaca aca ctc aac agc atg cac 720 Thr Leu Asp Asn Pro Leu Ser Lys Leu ThrThr Leu Asn Ser Met His 225 230 235 240 tcg cac ttc atc ctg tct gat gatggg acc gtg ggc aag tat gga aat 768 Ser His Phe Ile Leu Ser Asp Asp GlyThr Val Gly Lys Tyr Gly Asn 245 250 255 gaa atg aag ctc aga agg aac ctggag aag tac ctc tct ctg cag aaa 816 Glu Met Lys Leu Arg Arg Asn Leu GluLys Tyr Leu Ser Leu Gln Lys 260 265 270 ata cac tgc cgc tca aga caa ggcgtg ccg gtc gtg ggg ctg gtg gtg 864 Ile His Cys Arg Ser Arg Gln Gly ValPro Val Val Gly Leu Val Val 275 280 285 gaa ggc ggt ccc aac gtc atc ctgtca gtg tgg gag act gtc aag gac 912 Glu Gly Gly Pro Asn Val Ile Leu SerVal Trp Glu Thr Val Lys Asp 290 295 300 aag gac cca gtg gtg gtg tgt gagggc aca ggt agg gcg gct gac ctc 960 Lys Asp Pro Val Val Val Cys Glu GlyThr Gly Arg Ala Ala Asp Leu 305 310 315 320 ctg gcc ttc aca cac aaa cacctg gca gat gaa ggg atg ctg cga cct 1008 Leu Ala Phe Thr His Lys His LeuAla Asp Glu Gly Met Leu Arg Pro 325 330 335 cag gtg aaa gag gag atc atctgc atg att cag aac act ttc aac ttt 1056 Gln Val Lys Glu Glu Ile Ile CysMet Ile Gln Asn Thr Phe Asn Phe 340 345 350 agt ctt aaa cag tcc aag cacctt ttc caa att cta atg gag tgt atg 1104 Ser Leu Lys Gln Ser Lys His LeuPhe Gln Ile Leu Met Glu Cys Met 355 360 365 gtt cac agg gat tgt att accata ttt gat gct gac tct gaa gag cag 1152 Val His Arg Asp Cys Ile Thr IlePhe Asp Ala Asp Ser Glu Glu Gln 370 375 380 caa gac ctg gac tta gca atccta aca gct ttg ctg aag ggc aca aat 1200 Gln Asp Leu Asp Leu Ala Ile LeuThr Ala Leu Leu Lys Gly Thr Asn 385 390 395 400 tta tca gcg tca gag caatta aat ctg gca atg gct tgg gac agg gtg 1248 Leu Ser Ala Ser Glu Gln LeuAsn Leu Ala Met Ala Trp Asp Arg Val 405 410 415 gac att gcc aag aaa catatc cta att tat gaa caa cac tgg aag cct 1296 Asp Ile Ala Lys Lys His IleLeu Ile Tyr Glu Gln His Trp Lys Pro 420 425 430 gat gcc ctg gaa caa gcaatg tca gat gct tta gtg atg gat cgg gtg 1344 Asp Ala Leu Glu Gln Ala MetSer Asp Ala Leu Val Met Asp Arg Val 435 440 445 gat ttt gtg aag ctc ttaata gaa tat gga gtg aac ctc cat cgc ttt 1392 Asp Phe Val Lys Leu Leu IleGlu Tyr Gly Val Asn Leu His Arg Phe 450 455 460 ctt acc atc cct cga ctggaa gag ctc tac aat aca aaa caa gga cct 1440 Leu Thr Ile Pro Arg Leu GluGlu Leu Tyr Asn Thr Lys Gln Gly Pro 465 470 475 480 act aat aca ctc ttgcat cat ctc gtc caa gat gtg aaa cag cat acc 1488 Thr Asn Thr Leu Leu HisHis Leu Val Gln Asp Val Lys Gln His Thr 485 490 495 ctt ctt tca ggc taccga ata acc ttg att gac att gga tta gta gta 1536 Leu Leu Ser Gly Tyr ArgIle Thr Leu Ile Asp Ile Gly Leu Val Val 500 505 510 gaa tac ctc att ggtaga gca tat cgc agc aac tac act aga aaa cat 1584 Glu Tyr Leu Ile Gly ArgAla Tyr Arg Ser Asn Tyr Thr Arg Lys His 515 520 525 ttc aga gcc ctc tacaac aac ctc tac aga aaa tac aag cac cag aga 1632 Phe Arg Ala Leu Tyr AsnAsn Leu Tyr Arg Lys Tyr Lys His Gln Arg 530 535 540 cac tcc tca gga aataga aat gag tct gca gaa agt acg ctg cac tcc 1680 His Ser Ser Gly Asn ArgAsn Glu Ser Ala Glu Ser Thr Leu His Ser 545 550 555 560 cag ttc att agaact gca cag cca tac aaa ttc aag gaa aag tct ata 1728 Gln Phe Ile Arg ThrAla Gln Pro Tyr Lys Phe Lys Glu Lys Ser Ile 565 570 575 gtc ctt cat aaatca agg aag aag tca aaa gaa caa aat gta tca gat 1776 Val Leu His Lys SerArg Lys Lys Ser Lys Glu Gln Asn Val Ser Asp 580 585 590 gac cct gag tctact ggc ttt ctt tac cct tac aat gac ctg ctg gtt 1824 Asp Pro Glu Ser ThrGly Phe Leu Tyr Pro Tyr Asn Asp Leu Leu Val 595 600 605 tgg gct gtg ctgatg aaa agg cag aag atg gct atg ttc ttc tgg cag 1872 Trp Ala Val Leu MetLys Arg Gln Lys Met Ala Met Phe Phe Trp Gln 610 615 620 cat gga gag gaggcc acg gtt aaa gcc gtg att gcg tgt atc ctc tac 1920 His Gly Glu Glu AlaThr Val Lys Ala Val Ile Ala Cys Ile Leu Tyr 625 630 635 640 cgg gca atggcc cat gaa gct aag gag agt cac atg gtg gat gat gcc 1968 Arg Ala Met AlaHis Glu Ala Lys Glu Ser His Met Val Asp Asp Ala 645 650 655 tca gaa gagttg aag aat tac tca aaa cag ttt ggc cag ctg gct ctg 2016 Ser Glu Glu LeuLys Asn Tyr Ser Lys Gln Phe Gly Gln Leu Ala Leu 660 665 670 gac ttg ttggag aag gca ttc aag cag aat gag cgc atg gcc atg acg 2064 Asp Leu Leu GluLys Ala Phe Lys Gln Asn Glu Arg Met Ala Met Thr 675 680 685 ctg ttg acgtat gaa ctc agg aac tgg agc aat tcg acc tgc ctg aaa 2112 Leu Leu Thr TyrGlu Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys 690 695 700 ctg gcc gtgtcg gga gga tta cga ccc ttt gtt tca cat act tgt acc 2160 Leu Ala Val SerGly Gly Leu Arg Pro Phe Val Ser His Thr Cys Thr 705 710 715 720 cag atgcta ctg aca gac atg tgg atg ggg agg ctg aaa atg agg aaa 2208 Gln Met LeuLeu Thr Asp Met Trp Met Gly Arg Leu Lys Met Arg Lys 725 730 735 aac tcttgg tta aag att att ata agc att att tta cca ccc acc att 2256 Asn Ser TrpLeu Lys Ile Ile Ile Ser Ile Ile Leu Pro Pro Thr Ile 740 745 750 ttg acactg gaa ttt aaa agc aaa gct gag atg tca cat gtt ccc cag 2304 Leu Thr LeuGlu Phe Lys Ser Lys Ala Glu Met Ser His Val Pro Gln 755 760 765 tcc caggac ttc caa ttt atg tgg tat tac agt gac cag aac gcc agc 2352 Ser Gln AspPhe Gln Phe Met Trp Tyr Tyr Ser Asp Gln Asn Ala Ser 770 775 780 agt tccaaa gaa agt gct tct gtg aaa gag tat gat ttg gaa agg ggc 2400 Ser Ser LysGlu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu Arg Gly 785 790 795 800 catgat gag aaa ctg gat gaa aat cag cat ttt ggt ttg gaa agt ggg 2448 His AspGlu Lys Leu Asp Glu Asn Gln His Phe Gly Leu Glu Ser Gly 805 810 815 caccaa cac ctt ccg tgg acc agg aaa gtc tat gag ttc tac agt gct 2496 His GlnHis Leu Pro Trp Thr Arg Lys Val Tyr Glu Phe Tyr Ser Ala 820 825 830 ccaatt gtc aag ttt tgg ttt tat acg atg gcg tat ttg gca ttc ctc 2544 Pro IleVal Lys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala Phe Leu 835 840 845 atgctg ttc act tac acc gtg ttg gtg gag atg cag ccc cag ccc agc 2592 Met LeuPhe Thr Tyr Thr Val Leu Val Glu Met Gln Pro Gln Pro Ser 850 855 860 gtgcag gag tgg ctt gtt agc att tac atc ttc acc aat gct att gag 2640 Val GlnGlu Trp Leu Val Ser Ile Tyr Ile Phe Thr Asn Ala Ile Glu 865 870 875 880gtg gtc agg gag atc tgt att tca gaa cct ggg aag ttt acc caa aag 2688 ValVal Arg Glu Ile Cys Ile Ser Glu Pro Gly Lys Phe Thr Gln Lys 885 890 895gtg aag gta tgg att agt gag tac tgg aac tta aca gaa act gtg gcc 2736 ValLys Val Trp Ile Ser Glu Tyr Trp Asn Leu Thr Glu Thr Val Ala 900 905 910att ggc ctg ttt tca gct ggc ttc gtc ctt cga tgg ggt gac cct cct 2784 IleGly Leu Phe Ser Ala Gly Phe Val Leu Arg Trp Gly Asp Pro Pro 915 920 925ttt cac aca gcg gga aga ctg atc tac tgc ata gac atc ata ttc tgg 2832 PheHis Thr Ala Gly Arg Leu Ile Tyr Cys Ile Asp Ile Ile Phe Trp 930 935 940ttc tca cgg ctc ctg gac ttc ttt gct gtg aat caa cat gca ggt cca 2880 PheSer Arg Leu Leu Asp Phe Phe Ala Val Asn Gln His Ala Gly Pro 945 950 955960 tat gtg acc atg att gca aaa atg aca gca aac atg ttc tat att gtg 2928Tyr Val Thr Met Ile Ala Lys Met Thr Ala Asn Met Phe Tyr Ile Val 965 970975 atc atc atg gcc ata gtc ctg ctg agc ttt gga gtg gca cgc aag gcc 2976Ile Ile Met Ala Ile Val Leu Leu Ser Phe Gly Val Ala Arg Lys Ala 980 985990 atc ctt tcg cca aaa gag cca cca tct tgg agt cta gct cga gat att 3024Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp Ser Leu Ala Arg Asp Ile 995 10001005 gta ttt gag cca tac tgg atg ata tac gga gaa gtc tat gct gga 3069Val Phe Glu Pro Tyr Trp Met Ile Tyr Gly Glu Val Tyr Ala Gly 1010 10151020 gaa ata gat gtt tgt tca agc cag cca tcc tgc cct cct ggt tct 3114Glu Ile Asp Val Cys Ser Ser Gln Pro Ser Cys Pro Pro Gly Ser 1025 10301035 ttt ctt act cca ttc ttg caa gct gtc tac ctc ttc gtg caa tat 3159Phe Leu Thr Pro Phe Leu Gln Ala Val Tyr Leu Phe Val Gln Tyr 1040 10451050 atc atc atg gtg aac ctg ttg att gct ttc ttc aac aac gtt tac 3204Ile Ile Met Val Asn Leu Leu Ile Ala Phe Phe Asn Asn Val Tyr 1055 10601065 tta gat atg gaa tcc att tca aat aac ctg tgg aaa tac aac cgc 3249Leu Asp Met Glu Ser Ile Ser Asn Asn Leu Trp Lys Tyr Asn Arg 1070 10751080 tat cgc tac atc atg acc tac cac gag aag ccc tgg ctg ccc cca 3294Tyr Arg Tyr Ile Met Thr Tyr His Glu Lys Pro Trp Leu Pro Pro 1085 10901095 cct ctc atc ctg ctg agc cac gtg ggc ctt ctc ctc cgc cgc ctg 3339Pro Leu Ile Leu Leu Ser His Val Gly Leu Leu Leu Arg Arg Leu 1100 11051110 tgc tgt cat cga gct cct cac gac caa gaa gag ggt gac gtt gga 3384Cys Cys His Arg Ala Pro His Asp Gln Glu Glu Gly Asp Val Gly 1115 11201125 tta aaa ctc tac ctc agt aag gag gat ctg aaa aaa ctt cat gat 3429Leu Lys Leu Tyr Leu Ser Lys Glu Asp Leu Lys Lys Leu His Asp 1130 11351140 ttt gag gag cag tgc gtg gaa aaa tac ttc cat gag aag atg gaa 3474Phe Glu Glu Gln Cys Val Glu Lys Tyr Phe His Glu Lys Met Glu 1145 11501155 gat gtg aat tgt agt tgt gag gaa cga atc cga gtg aca tca gaa 3519Asp Val Asn Cys Ser Cys Glu Glu Arg Ile Arg Val Thr Ser Glu 1160 11651170 agg gtt aca gag atg tac ttc cag ctg aaa gaa atg aat gaa aag 3564Arg Val Thr Glu Met Tyr Phe Gln Leu Lys Glu Met Asn Glu Lys 1175 11801185 gtg tct ttt ata aag gac tcc tta ctg tct ttg gac agc cag gtg 3609Val Ser Phe Ile Lys Asp Ser Leu Leu Ser Leu Asp Ser Gln Val 1190 11951200 gga cac ctg cag gat ctc tct gcc ctg act gtg gat acc ctg aaa 3654Gly His Leu Gln Asp Leu Ser Ala Leu Thr Val Asp Thr Leu Lys 1205 12101215 gtc ctt tct gct gtt gac act ttg caa gag gat gag gct ctc ctg 3699Val Leu Ser Ala Val Asp Thr Leu Gln Glu Asp Glu Ala Leu Leu 1220 12251230 gcc aag aga aag cat tct act tgc aaa aaa ctt ccc cac agc tgg 3744Ala Lys Arg Lys His Ser Thr Cys Lys Lys Leu Pro His Ser Trp 1235 12401245 agc aat gtc atc tgt gca gag gtt cta ggc agc atg gag atc gct 3789Ser Asn Val Ile Cys Ala Glu Val Leu Gly Ser Met Glu Ile Ala 1250 12551260 gga gag aag aaa tac cag tat tat agc atg ccc tct tct ttg ctg 3834Gly Glu Lys Lys Tyr Gln Tyr Tyr Ser Met Pro Ser Ser Leu Leu 1265 12701275 agg agc ctg gct gga ggc cgg cat ccc cca aga gtg cag agg ggg 3879Arg Ser Leu Ala Gly Gly Arg His Pro Pro Arg Val Gln Arg Gly 1280 12851290 gca ctt ctt gag att aca aac agt aaa aga gag gct aca aat gta 3924Ala Leu Leu Glu Ile Thr Asn Ser Lys Arg Glu Ala Thr Asn Val 1295 13001305 aga aat gac cag gaa agg caa gaa aca caa agt agt ata gtg gtt 3969Arg Asn Asp Gln Glu Arg Gln Glu Thr Gln Ser Ser Ile Val Val 1310 13151320 tct ggg gtg tct cct aac agg caa gca cac tca aag tat ggc cag 4014Ser Gly Val Ser Pro Asn Arg Gln Ala His Ser Lys Tyr Gly Gln 1325 13301335 ttt ctt ctg gtc ccc tct aat cta aag cga gtt cct ttt tca gca 4059Phe Leu Leu Val Pro Ser Asn Leu Lys Arg Val Pro Phe Ser Ala 1340 13451350 gaa act gtc ttg cct ctg tcc aga ccc tct gtg cca gat gtg ctg 4104Glu Thr Val Leu Pro Leu Ser Arg Pro Ser Val Pro Asp Val Leu 1355 13601365 gca act gaa cag gac atc cag act gag gtt ctt gtt cat ctg act 4149Ala Thr Glu Gln Asp Ile Gln Thr Glu Val Leu Val His Leu Thr 1370 13751380 ggg cag acc cca gtt gtc tct gac tgg gca tca gtg gat gaa ccc 4194Gly Gln Thr Pro Val Val Ser Asp Trp Ala Ser Val Asp Glu Pro 1385 13901395 aag gaa aag cac gag cct att gct cac tta ctg gat gga caa gac 4239Lys Glu Lys His Glu Pro Ile Ala His Leu Leu Asp Gly Gln Asp 1400 14051410 aag gca gag caa gtg cta ccc act ttg agt tgc aca cct gaa ccc 4284Lys Ala Glu Gln Val Leu Pro Thr Leu Ser Cys Thr Pro Glu Pro 1415 14201425 atg aca atg agc tcc cct ctt tcc caa gcc aag atc atg caa act 4329Met Thr Met Ser Ser Pro Leu Ser Gln Ala Lys Ile Met Gln Thr 1430 14351440 gga ggt gga tat gta aac tgg gca ttt tca gaa ggt gat gaa act 4374Gly Gly Gly Tyr Val Asn Trp Ala Phe Ser Glu Gly Asp Glu Thr 1445 14501455 ggt gtg ttt agc atc aag aaa aag tgg caa acc tgc ttg ccc tcc 4419Gly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr Cys Leu Pro Ser 1460 14651470 act tgt gac agt gat tcc tct cgg agt gaa cag cac cag aag cag 4464Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln Lys Gln 1475 14801485 gcc cag gac agc tcc cta tct gat aac tca aca aga tcg gcc cag 4509Ala Gln Asp Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln 1490 14951500 agt agt gaa tgc tca gag gtg gga cca tgg ctt cag cca aac aca 4554Ser Ser Glu Cys Ser Glu Val Gly Pro Trp Leu Gln Pro Asn Thr 1505 15101515 tcc ttt tgg atc aat cct ctc cgc aga tac agg ccc ttc gct agg 4599Ser Phe Trp Ile Asn Pro Leu Arg Arg Tyr Arg Pro Phe Ala Arg 1520 15251530 agt cat agt ttt aga ttc cat aag gag gag aaa ttg atg aag atc 4644Ser His Ser Phe Arg Phe His Lys Glu Glu Lys Leu Met Lys Ile 1535 15401545 tgt aag att aaa aat ctt tca ggc tct tca gaa ata ggg cag gga 4689Cys Lys Ile Lys Asn Leu Ser Gly Ser Ser Glu Ile Gly Gln Gly 1550 15551560 gca tgg gtc aaa gcg aaa atg cta acc aaa gac agg aga ctg tca 4734Ala Trp Val Lys Ala Lys Met Leu Thr Lys Asp Arg Arg Leu Ser 1565 15701575 aag aaa aag aag aat act caa gga ctc cag gtg cca atc ata aca 4779Lys Lys Lys Lys Asn Thr Gln Gly Leu Gln Val Pro Ile Ile Thr 1580 15851590 gtc aat gcc tgc tct cag agt gac cag ttg aat cca gag cca gga 4824Val Asn Ala Cys Ser Gln Ser Asp Gln Leu Asn Pro Glu Pro Gly 1595 16001605 gaa aac agc atc tct gaa gag gag tac agc aag aac tgg ttc aca 4869Glu Asn Ser Ile Ser Glu Glu Glu Tyr Ser Lys Asn Trp Phe Thr 1610 16151620 gtg tcc aaa ttt agt cac aca ggt gta gaa cct tac ata cat cag 4914Val Ser Lys Phe Ser His Thr Gly Val Glu Pro Tyr Ile His Gln 1625 16301635 aaa atg aaa act aaa gaa att gga caa tgt gct ata caa atc agt 4959Lys Met Lys Thr Lys Glu Ile Gly Gln Cys Ala Ile Gln Ile Ser 1640 16451650 gat tac cta aag cag tct caa gag gat ctc agc aaa aac tct ttg 5004Asp Tyr Leu Lys Gln Ser Gln Glu Asp Leu Ser Lys Asn Ser Leu 1655 16601665 tgg aat tcc agg agc acc aac ctc aat agg aac tcc ctg ctg aaa 5049Trp Asn Ser Arg Ser Thr Asn Leu Asn Arg Asn Ser Leu Leu Lys 1670 16751680 agt tca att gga gtt gac aag atc tca gcc tcc tta aaa agc cct 5094Ser Ser Ile Gly Val Asp Lys Ile Ser Ala Ser Leu Lys Ser Pro 1685 16901695 caa gag cct cac cat cat tat tca gcc att gaa agg aat aat tta 5139Gln Glu Pro His His His Tyr Ser Ala Ile Glu Arg Asn Asn Leu 1700 17051710 atg agg ctt tct cag acc ata cca ttt aca cca gtc caa ctg ttt 5184Met Arg Leu Ser Gln Thr Ile Pro Phe Thr Pro Val Gln Leu Phe 1715 17201725 gca gga gaa gaa ata act gtc tac agg ttg gag gag agt tcc cct 5229Ala Gly Glu Glu Ile Thr Val Tyr Arg Leu Glu Glu Ser Ser Pro 1730 17351740 tta aac ctt gat aaa agc atg tcc tct tgg tct cag cgt ggg aga 5274Leu Asn Leu Asp Lys Ser Met Ser Ser Trp Ser Gln Arg Gly Arg 1745 17501755 gcg gca atg atc cag gta ttg tcc cga gag gag atg gat ggg ggc 5319Ala Ala Met Ile Gln Val Leu Ser Arg Glu Glu Met Asp Gly Gly 1760 17651770 ctc cgt aaa gct atg aga gtc gtc agc act tgg tct gag gat gac 5364Leu Arg Lys Ala Met Arg Val Val Ser Thr Trp Ser Glu Asp Asp 1775 17801785 att ctc aag ccg gga caa gtt ttc att gtc aag tcc ttt ctt cct 5409Ile Leu Lys Pro Gly Gln Val Phe Ile Val Lys Ser Phe Leu Pro 1790 17951800 gag gtt gtg cgg aca tgg cat aaa atc ttc cag gag agc act gtg 5454Glu Val Val Arg Thr Trp His Lys Ile Phe Gln Glu Ser Thr Val 1805 18101815 ctt cat ctt tgc ctc agg gaa att caa caa caa aga gct gct caa 5499Leu His Leu Cys Leu Arg Glu Ile Gln Gln Gln Arg Ala Ala Gln 1820 18251830 aaa ttg atc tat acc ttc aac caa gtg aaa cca caa acc ata ccc 5544Lys Leu Ile Tyr Thr Phe Asn Gln Val Lys Pro Gln Thr Ile Pro 1835 18401845 tac aca cca agg ttc ctg gaa gtt ttc tta atc tac tgc cat tca 5589Tyr Thr Pro Arg Phe Leu Glu Val Phe Leu Ile Tyr Cys His Ser 1850 18551860 gcc aac cag tgg ttg acc att gag aag tat atg aca ggg gag ttc 5634Ala Asn Gln Trp Leu Thr Ile Glu Lys Tyr Met Thr Gly Glu Phe 1865 18701875 cgg aag tat aac aac aac aat ggt gat gaa atc acc ccc acc aac 5679Arg Lys Tyr Asn Asn Asn Asn Gly Asp Glu Ile Thr Pro Thr Asn 1880 18851890 acc ctg gag gag ctg atg ttg gct ttc tct cac tgg acc tat gag 5724Thr Leu Glu Glu Leu Met Leu Ala Phe Ser His Trp Thr Tyr Glu 1895 19001905 tac act cgg gga gag ctg ctg gtt tta gat ttg caa ggt gtt gga 5769Tyr Thr Arg Gly Glu Leu Leu Val Leu Asp Leu Gln Gly Val Gly 1910 19151920 gaa aat ttg aca gat cca tct gtt ata aaa cct gaa gtc aaa caa 5814Glu Asn Leu Thr Asp Pro Ser Val Ile Lys Pro Glu Val Lys Gln 1925 19301935 tca aga gga atg gtg ttt gga ccg gcc aat ttg ggg gaa gat gca 5859Ser Arg Gly Met Val Phe Gly Pro Ala Asn Leu Gly Glu Asp Ala 1940 19451950 att aga aac ttc att gca aaa cat cat tgt aac tcc tgc tgc cgg 5904Ile Arg Asn Phe Ile Ala Lys His His Cys Asn Ser Cys Cys Arg 1955 19601965 aag ctc aaa ctc ccg gat tta aaa aga aat gac tat tcc cct gaa 5949Lys Leu Lys Leu Pro Asp Leu Lys Arg Asn Asp Tyr Ser Pro Glu 1970 19751980 agg ata aat tcc acc ttt gga ctt gag ata aaa ata gaa tca gct 5994Arg Ile Asn Ser Thr Phe Gly Leu Glu Ile Lys Ile Glu Ser Ala 1985 19901995 gag gag cct cca gca agg gag acg ggt aga aat tcc cca gaa gat 6039Glu Glu Pro Pro Ala Arg Glu Thr Gly Arg Asn Ser Pro Glu Asp 2000 20052010 gat atg caa cta taa 6054 Asp Met Gln Leu 2015 2 2017 PRT Homosapiens 2 Met Ile Ile Leu Ser Lys Ser Gln Lys Ser Trp Ile Lys Gly ValPhe 1 5 10 15 Asp Lys Arg Glu Cys Ser Thr Ile Ile Pro Ser Ser Lys AsnPro His 20 25 30 Arg Cys Thr Pro Val Cys Gln Val Cys Gln Asn Leu Ile ArgCys Tyr 35 40 45 Cys Gly Arg Leu Ile Gly Asp His Ala Gly Ile Asp Tyr SerTrp Thr 50 55 60 Ile Ser Ala Ala Lys Gly Lys Glu Ser Glu Gln Trp Ser ValGlu Lys 65 70 75 80 His Thr Thr Lys Ser Pro Thr Asp Thr Phe Gly Thr IleAsn Phe Gln 85 90 95 Asp Gly Glu His Thr His His Ala Lys Tyr Ile Arg ThrSer Tyr Asp 100 105 110 Thr Lys Leu Asp His Leu Leu His Leu Met Leu LysGlu Trp Lys Met 115 120 125 Glu Leu Pro Lys Leu Val Ile Ser Val His GlyGly Ile Gln Asn Phe 130 135 140 Thr Met Pro Ser Lys Phe Lys Glu Ile PheSer Gln Gly Leu Val Lys 145 150 155 160 Ala Ala Glu Thr Thr Gly Ala TrpIle Ile Thr Glu Gly Ile Asn Thr 165 170 175 Gly Val Ser Lys His Val GlyAsp Ala Leu Lys Ser His Ser Ser His 180 185 190 Ser Leu Arg Lys Ile TrpThr Val Gly Ile Pro Pro Trp Gly Val Ile 195 200 205 Glu Asn Gln Arg AspLeu Ile Gly Lys Asp Val Val Cys Leu Tyr Gln 210 215 220 Thr Leu Asp AsnPro Leu Ser Lys Leu Thr Thr Leu Asn Ser Met His 225 230 235 240 Ser HisPhe Ile Leu Ser Asp Asp Gly Thr Val Gly Lys Tyr Gly Asn 245 250 255 GluMet Lys Leu Arg Arg Asn Leu Glu Lys Tyr Leu Ser Leu Gln Lys 260 265 270Ile His Cys Arg Ser Arg Gln Gly Val Pro Val Val Gly Leu Val Val 275 280285 Glu Gly Gly Pro Asn Val Ile Leu Ser Val Trp Glu Thr Val Lys Asp 290295 300 Lys Asp Pro Val Val Val Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu305 310 315 320 Leu Ala Phe Thr His Lys His Leu Ala Asp Glu Gly Met LeuArg Pro 325 330 335 Gln Val Lys Glu Glu Ile Ile Cys Met Ile Gln Asn ThrPhe Asn Phe 340 345 350 Ser Leu Lys Gln Ser Lys His Leu Phe Gln Ile LeuMet Glu Cys Met 355 360 365 Val His Arg Asp Cys Ile Thr Ile Phe Asp AlaAsp Ser Glu Glu Gln 370 375 380 Gln Asp Leu Asp Leu Ala Ile Leu Thr AlaLeu Leu Lys Gly Thr Asn 385 390 395 400 Leu Ser Ala Ser Glu Gln Leu AsnLeu Ala Met Ala Trp Asp Arg Val 405 410 415 Asp Ile Ala Lys Lys His IleLeu Ile Tyr Glu Gln His Trp Lys Pro 420 425 430 Asp Ala Leu Glu Gln AlaMet Ser Asp Ala Leu Val Met Asp Arg Val 435 440 445 Asp Phe Val Lys LeuLeu Ile Glu Tyr Gly Val Asn Leu His Arg Phe 450 455 460 Leu Thr Ile ProArg Leu Glu Glu Leu Tyr Asn Thr Lys Gln Gly Pro 465 470 475 480 Thr AsnThr Leu Leu His His Leu Val Gln Asp Val Lys Gln His Thr 485 490 495 LeuLeu Ser Gly Tyr Arg Ile Thr Leu Ile Asp Ile Gly Leu Val Val 500 505 510Glu Tyr Leu Ile Gly Arg Ala Tyr Arg Ser Asn Tyr Thr Arg Lys His 515 520525 Phe Arg Ala Leu Tyr Asn Asn Leu Tyr Arg Lys Tyr Lys His Gln Arg 530535 540 His Ser Ser Gly Asn Arg Asn Glu Ser Ala Glu Ser Thr Leu His Ser545 550 555 560 Gln Phe Ile Arg Thr Ala Gln Pro Tyr Lys Phe Lys Glu LysSer Ile 565 570 575 Val Leu His Lys Ser Arg Lys Lys Ser Lys Glu Gln AsnVal Ser Asp 580 585 590 Asp Pro Glu Ser Thr Gly Phe Leu Tyr Pro Tyr AsnAsp Leu Leu Val 595 600 605 Trp Ala Val Leu Met Lys Arg Gln Lys Met AlaMet Phe Phe Trp Gln 610 615 620 His Gly Glu Glu Ala Thr Val Lys Ala ValIle Ala Cys Ile Leu Tyr 625 630 635 640 Arg Ala Met Ala His Glu Ala LysGlu Ser His Met Val Asp Asp Ala 645 650 655 Ser Glu Glu Leu Lys Asn TyrSer Lys Gln Phe Gly Gln Leu Ala Leu 660 665 670 Asp Leu Leu Glu Lys AlaPhe Lys Gln Asn Glu Arg Met Ala Met Thr 675 680 685 Leu Leu Thr Tyr GluLeu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys 690 695 700 Leu Ala Val SerGly Gly Leu Arg Pro Phe Val Ser His Thr Cys Thr 705 710 715 720 Gln MetLeu Leu Thr Asp Met Trp Met Gly Arg Leu Lys Met Arg Lys 725 730 735 AsnSer Trp Leu Lys Ile Ile Ile Ser Ile Ile Leu Pro Pro Thr Ile 740 745 750Leu Thr Leu Glu Phe Lys Ser Lys Ala Glu Met Ser His Val Pro Gln 755 760765 Ser Gln Asp Phe Gln Phe Met Trp Tyr Tyr Ser Asp Gln Asn Ala Ser 770775 780 Ser Ser Lys Glu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu Arg Gly785 790 795 800 His Asp Glu Lys Leu Asp Glu Asn Gln His Phe Gly Leu GluSer Gly 805 810 815 His Gln His Leu Pro Trp Thr Arg Lys Val Tyr Glu PheTyr Ser Ala 820 825 830 Pro Ile Val Lys Phe Trp Phe Tyr Thr Met Ala TyrLeu Ala Phe Leu 835 840 845 Met Leu Phe Thr Tyr Thr Val Leu Val Glu MetGln Pro Gln Pro Ser 850 855 860 Val Gln Glu Trp Leu Val Ser Ile Tyr IlePhe Thr Asn Ala Ile Glu 865 870 875 880 Val Val Arg Glu Ile Cys Ile SerGlu Pro Gly Lys Phe Thr Gln Lys 885 890 895 Val Lys Val Trp Ile Ser GluTyr Trp Asn Leu Thr Glu Thr Val Ala 900 905 910 Ile Gly Leu Phe Ser AlaGly Phe Val Leu Arg Trp Gly Asp Pro Pro 915 920 925 Phe His Thr Ala GlyArg Leu Ile Tyr Cys Ile Asp Ile Ile Phe Trp 930 935 940 Phe Ser Arg LeuLeu Asp Phe Phe Ala Val Asn Gln His Ala Gly Pro 945 950 955 960 Tyr ValThr Met Ile Ala Lys Met Thr Ala Asn Met Phe Tyr Ile Val 965 970 975 IleIle Met Ala Ile Val Leu Leu Ser Phe Gly Val Ala Arg Lys Ala 980 985 990Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp Ser Leu Ala Arg Asp Ile 995 10001005 Val Phe Glu Pro Tyr Trp Met Ile Tyr Gly Glu Val Tyr Ala Gly 10101015 1020 Glu Ile Asp Val Cys Ser Ser Gln Pro Ser Cys Pro Pro Gly Ser1025 1030 1035 Phe Leu Thr Pro Phe Leu Gln Ala Val Tyr Leu Phe Val GlnTyr 1040 1045 1050 Ile Ile Met Val Asn Leu Leu Ile Ala Phe Phe Asn AsnVal Tyr 1055 1060 1065 Leu Asp Met Glu Ser Ile Ser Asn Asn Leu Trp LysTyr Asn Arg 1070 1075 1080 Tyr Arg Tyr Ile Met Thr Tyr His Glu Lys ProTrp Leu Pro Pro 1085 1090 1095 Pro Leu Ile Leu Leu Ser His Val Gly LeuLeu Leu Arg Arg Leu 1100 1105 1110 Cys Cys His Arg Ala Pro His Asp GlnGlu Glu Gly Asp Val Gly 1115 1120 1125 Leu Lys Leu Tyr Leu Ser Lys GluAsp Leu Lys Lys Leu His Asp 1130 1135 1140 Phe Glu Glu Gln Cys Val GluLys Tyr Phe His Glu Lys Met Glu 1145 1150 1155 Asp Val Asn Cys Ser CysGlu Glu Arg Ile Arg Val Thr Ser Glu 1160 1165 1170 Arg Val Thr Glu MetTyr Phe Gln Leu Lys Glu Met Asn Glu Lys 1175 1180 1185 Val Ser Phe IleLys Asp Ser Leu Leu Ser Leu Asp Ser Gln Val 1190 1195 1200 Gly His LeuGln Asp Leu Ser Ala Leu Thr Val Asp Thr Leu Lys 1205 1210 1215 Val LeuSer Ala Val Asp Thr Leu Gln Glu Asp Glu Ala Leu Leu 1220 1225 1230 AlaLys Arg Lys His Ser Thr Cys Lys Lys Leu Pro His Ser Trp 1235 1240 1245Ser Asn Val Ile Cys Ala Glu Val Leu Gly Ser Met Glu Ile Ala 1250 12551260 Gly Glu Lys Lys Tyr Gln Tyr Tyr Ser Met Pro Ser Ser Leu Leu 12651270 1275 Arg Ser Leu Ala Gly Gly Arg His Pro Pro Arg Val Gln Arg Gly1280 1285 1290 Ala Leu Leu Glu Ile Thr Asn Ser Lys Arg Glu Ala Thr AsnVal 1295 1300 1305 Arg Asn Asp Gln Glu Arg Gln Glu Thr Gln Ser Ser IleVal Val 1310 1315 1320 Ser Gly Val Ser Pro Asn Arg Gln Ala His Ser LysTyr Gly Gln 1325 1330 1335 Phe Leu Leu Val Pro Ser Asn Leu Lys Arg ValPro Phe Ser Ala 1340 1345 1350 Glu Thr Val Leu Pro Leu Ser Arg Pro SerVal Pro Asp Val Leu 1355 1360 1365 Ala Thr Glu Gln Asp Ile Gln Thr GluVal Leu Val His Leu Thr 1370 1375 1380 Gly Gln Thr Pro Val Val Ser AspTrp Ala Ser Val Asp Glu Pro 1385 1390 1395 Lys Glu Lys His Glu Pro IleAla His Leu Leu Asp Gly Gln Asp 1400 1405 1410 Lys Ala Glu Gln Val LeuPro Thr Leu Ser Cys Thr Pro Glu Pro 1415 1420 1425 Met Thr Met Ser SerPro Leu Ser Gln Ala Lys Ile Met Gln Thr 1430 1435 1440 Gly Gly Gly TyrVal Asn Trp Ala Phe Ser Glu Gly Asp Glu Thr 1445 1450 1455 Gly Val PheSer Ile Lys Lys Lys Trp Gln Thr Cys Leu Pro Ser 1460 1465 1470 Thr CysAsp Ser Asp Ser Ser Arg Ser Glu Gln His Gln Lys Gln 1475 1480 1485 AlaGln Asp Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln 1490 1495 1500Ser Ser Glu Cys Ser Glu Val Gly Pro Trp Leu Gln Pro Asn Thr 1505 15101515 Ser Phe Trp Ile Asn Pro Leu Arg Arg Tyr Arg Pro Phe Ala Arg 15201525 1530 Ser His Ser Phe Arg Phe His Lys Glu Glu Lys Leu Met Lys Ile1535 1540 1545 Cys Lys Ile Lys Asn Leu Ser Gly Ser Ser Glu Ile Gly GlnGly 1550 1555 1560 Ala Trp Val Lys Ala Lys Met Leu Thr Lys Asp Arg ArgLeu Ser 1565 1570 1575 Lys Lys Lys Lys Asn Thr Gln Gly Leu Gln Val ProIle Ile Thr 1580 1585 1590 Val Asn Ala Cys Ser Gln Ser Asp Gln Leu AsnPro Glu Pro Gly 1595 1600 1605 Glu Asn Ser Ile Ser Glu Glu Glu Tyr SerLys Asn Trp Phe Thr 1610 1615 1620 Val Ser Lys Phe Ser His Thr Gly ValGlu Pro Tyr Ile His Gln 1625 1630 1635 Lys Met Lys Thr Lys Glu Ile GlyGln Cys Ala Ile Gln Ile Ser 1640 1645 1650 Asp Tyr Leu Lys Gln Ser GlnGlu Asp Leu Ser Lys Asn Ser Leu 1655 1660 1665 Trp Asn Ser Arg Ser ThrAsn Leu Asn Arg Asn Ser Leu Leu Lys 1670 1675 1680 Ser Ser Ile Gly ValAsp Lys Ile Ser Ala Ser Leu Lys Ser Pro 1685 1690 1695 Gln Glu Pro HisHis His Tyr Ser Ala Ile Glu Arg Asn Asn Leu 1700 1705 1710 Met Arg LeuSer Gln Thr Ile Pro Phe Thr Pro Val Gln Leu Phe 1715 1720 1725 Ala GlyGlu Glu Ile Thr Val Tyr Arg Leu Glu Glu Ser Ser Pro 1730 1735 1740 LeuAsn Leu Asp Lys Ser Met Ser Ser Trp Ser Gln Arg Gly Arg 1745 1750 1755Ala Ala Met Ile Gln Val Leu Ser Arg Glu Glu Met Asp Gly Gly 1760 17651770 Leu Arg Lys Ala Met Arg Val Val Ser Thr Trp Ser Glu Asp Asp 17751780 1785 Ile Leu Lys Pro Gly Gln Val Phe Ile Val Lys Ser Phe Leu Pro1790 1795 1800 Glu Val Val Arg Thr Trp His Lys Ile Phe Gln Glu Ser ThrVal 1805 1810 1815 Leu His Leu Cys Leu Arg Glu Ile Gln Gln Gln Arg AlaAla Gln 1820 1825 1830 Lys Leu Ile Tyr Thr Phe Asn Gln Val Lys Pro GlnThr Ile Pro 1835 1840 1845 Tyr Thr Pro Arg Phe Leu Glu Val Phe Leu IleTyr Cys His Ser 1850 1855 1860 Ala Asn Gln Trp Leu Thr Ile Glu Lys TyrMet Thr Gly Glu Phe 1865 1870 1875 Arg Lys Tyr Asn Asn Asn Asn Gly AspGlu Ile Thr Pro Thr Asn 1880 1885 1890 Thr Leu Glu Glu Leu Met Leu AlaPhe Ser His Trp Thr Tyr Glu 1895 1900 1905 Tyr Thr Arg Gly Glu Leu LeuVal Leu Asp Leu Gln Gly Val Gly 1910 1915 1920 Glu Asn Leu Thr Asp ProSer Val Ile Lys Pro Glu Val Lys Gln 1925 1930 1935 Ser Arg Gly Met ValPhe Gly Pro Ala Asn Leu Gly Glu Asp Ala 1940 1945 1950 Ile Arg Asn PheIle Ala Lys His His Cys Asn Ser Cys Cys Arg 1955 1960 1965 Lys Leu LysLeu Pro Asp Leu Lys Arg Asn Asp Tyr Ser Pro Glu 1970 1975 1980 Arg IleAsn Ser Thr Phe Gly Leu Glu Ile Lys Ile Glu Ser Ala 1985 1990 1995 GluGlu Pro Pro Ala Arg Glu Thr Gly Arg Asn Ser Pro Glu Asp 2000 2005 2010Asp Met Gln Leu 2015 3 5913 DNA Homo sapiens CDS (1)..(5910) 3 atg attatc cta tct aag tcc cag aaa tcc tgg att aaa gga gta ttt 48 Met Ile IleLeu Ser Lys Ser Gln Lys Ser Trp Ile Lys Gly Val Phe 1 5 10 15 gac aagaga gaa tgt agc aca atc ata ccc agc tca aaa aat cct cac 96 Asp Lys ArgGlu Cys Ser Thr Ile Ile Pro Ser Ser Lys Asn Pro His 20 25 30 aga tgt actcca gta tgc caa gtc tgc cag aat tta atc agg tgt tac 144 Arg Cys Thr ProVal Cys Gln Val Cys Gln Asn Leu Ile Arg Cys Tyr 35 40 45 tgt ggc cga ctgatt gga gac cat gct ggg ata gat tat tcc tgg acc 192 Cys Gly Arg Leu IleGly Asp His Ala Gly Ile Asp Tyr Ser Trp Thr 50 55 60 atc tca gct gcc aagggt aaa gaa agt gaa caa tgg tct gtt gaa aag 240 Ile Ser Ala Ala Lys GlyLys Glu Ser Glu Gln Trp Ser Val Glu Lys 65 70 75 80 cac aca acg aaa agccca aca gat act ttt ggc acg att aat ttc caa 288 His Thr Thr Lys Ser ProThr Asp Thr Phe Gly Thr Ile Asn Phe Gln 85 90 95 gat gga gag cac acc catcat gcc aag tat att aga act tct tat gat 336 Asp Gly Glu His Thr His HisAla Lys Tyr Ile Arg Thr Ser Tyr Asp 100 105 110 aca aaa ctg gat cat ctgtta cat tta atg ttg aaa gag tgg aaa atg 384 Thr Lys Leu Asp His Leu LeuHis Leu Met Leu Lys Glu Trp Lys Met 115 120 125 gaa ctg ccc aag ctt gtgatc tca gtc cat ggg ggc atc cag aac ttt 432 Glu Leu Pro Lys Leu Val IleSer Val His Gly Gly Ile Gln Asn Phe 130 135 140 act atg ccc tct aaa tttaaa gag att ttc agc caa ggt ttg gtt aaa 480 Thr Met Pro Ser Lys Phe LysGlu Ile Phe Ser Gln Gly Leu Val Lys 145 150 155 160 gct gca gag aca acagga gcg tgg ata ata act gaa ggc atc aat aca 528 Ala Ala Glu Thr Thr GlyAla Trp Ile Ile Thr Glu Gly Ile Asn Thr 165 170 175 gga gtg tcc aag catgtt ggg gat gcc ttg aaa tcc cat tcc tct cat 576 Gly Val Ser Lys His ValGly Asp Ala Leu Lys Ser His Ser Ser His 180 185 190 tcc ttg aga aaa atctgg aca gtt gga atc cct cct tgg ggt gtc att 624 Ser Leu Arg Lys Ile TrpThr Val Gly Ile Pro Pro Trp Gly Val Ile 195 200 205 gag aac cag aga gacctt att gga aaa gat gtg gtg tgc ctg tac cag 672 Glu Asn Gln Arg Asp LeuIle Gly Lys Asp Val Val Cys Leu Tyr Gln 210 215 220 act ctg gat aac cccctc agc aag ctc aca aca ctc aac agc atg cac 720 Thr Leu Asp Asn Pro LeuSer Lys Leu Thr Thr Leu Asn Ser Met His 225 230 235 240 tcg cac ttc atcctg tct gat gat ggg acc gtg ggc aag tat gga aat 768 Ser His Phe Ile LeuSer Asp Asp Gly Thr Val Gly Lys Tyr Gly Asn 245 250 255 gaa atg aag ctcaga agg aac ctg gag aag tac ctc tct ctg cag aaa 816 Glu Met Lys Leu ArgArg Asn Leu Glu Lys Tyr Leu Ser Leu Gln Lys 260 265 270 ata cac tgc cgctca aga caa ggc gtg ccg gtc gtg ggg ctg gtg gtg 864 Ile His Cys Arg SerArg Gln Gly Val Pro Val Val Gly Leu Val Val 275 280 285 gaa ggc ggt cccaac gtc atc ctg tca gtg tgg gag act gtc aag gac 912 Glu Gly Gly Pro AsnVal Ile Leu Ser Val Trp Glu Thr Val Lys Asp 290 295 300 aag gac cca gtggtg gtg tgt gag ggc aca ggt agg gcg gct gac ctc 960 Lys Asp Pro Val ValVal Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu 305 310 315 320 ctg gcc ttcaca cac aaa cac ctg gca gat gaa ggg atg ctg cga cct 1008 Leu Ala Phe ThrHis Lys His Leu Ala Asp Glu Gly Met Leu Arg Pro 325 330 335 cag gtg aaagag gag atc atc tgc atg att cag aac act ttc aac ttt 1056 Gln Val Lys GluGlu Ile Ile Cys Met Ile Gln Asn Thr Phe Asn Phe 340 345 350 agt ctt aaacag tcc aag cac ctt ttc caa att cta atg gag tgt atg 1104 Ser Leu Lys GlnSer Lys His Leu Phe Gln Ile Leu Met Glu Cys Met 355 360 365 gtt cac agggat tgt att acc ata ttt gat gct gac tct gaa gag cag 1152 Val His Arg AspCys Ile Thr Ile Phe Asp Ala Asp Ser Glu Glu Gln 370 375 380 caa gac ctggac tta gca atc cta aca gct ttg ctg aag ggc aca aat 1200 Gln Asp Leu AspLeu Ala Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn 385 390 395 400 tta tcagcg tca gag caa tta aat ctg gca atg gct tgg gac agg gtg 1248 Leu Ser AlaSer Glu Gln Leu Asn Leu Ala Met Ala Trp Asp Arg Val 405 410 415 gac attgcc aag aaa cat atc cta att tat gaa caa cac tgg aag cct 1296 Asp Ile AlaLys Lys His Ile Leu Ile Tyr Glu Gln His Trp Lys Pro 420 425 430 gat gccctg gaa caa gca atg tca gat gct tta gtg atg gat cgg gtg 1344 Asp Ala LeuGlu Gln Ala Met Ser Asp Ala Leu Val Met Asp Arg Val 435 440 445 gat tttgtg aag ctc tta ata gaa tat gga gtg aac ctc cat cgc ttt 1392 Asp Phe ValLys Leu Leu Ile Glu Tyr Gly Val Asn Leu His Arg Phe 450 455 460 ctt accatc cct cga ctg gaa gag ctc tac aat aca aaa caa gga cct 1440 Leu Thr IlePro Arg Leu Glu Glu Leu Tyr Asn Thr Lys Gln Gly Pro 465 470 475 480 actaat aca ctc ttg cat cat ctc gtc caa gat gtg aaa cag cac cag 1488 Thr AsnThr Leu Leu His His Leu Val Gln Asp Val Lys Gln His Gln 485 490 495 agacac tcc tca gga aat aga aat gag tct gca gaa agt acg ctg cac 1536 Arg HisSer Ser Gly Asn Arg Asn Glu Ser Ala Glu Ser Thr Leu His 500 505 510 tcccag ttc att aga act gca cag cca tac aaa ttc aag gaa aag tct 1584 Ser GlnPhe Ile Arg Thr Ala Gln Pro Tyr Lys Phe Lys Glu Lys Ser 515 520 525 atagtc ctt cat aaa tca agg aag aag tca aaa gaa caa aat gta tca 1632 Ile ValLeu His Lys Ser Arg Lys Lys Ser Lys Glu Gln Asn Val Ser 530 535 540 gatgac cct gag tct act ggc ttt ctt tac cct tac aat gac ctg ctg 1680 Asp AspPro Glu Ser Thr Gly Phe Leu Tyr Pro Tyr Asn Asp Leu Leu 545 550 555 560gtt tgg gct gtg ctg atg aaa agg cag aag atg gct atg ttc ttc tgg 1728 ValTrp Ala Val Leu Met Lys Arg Gln Lys Met Ala Met Phe Phe Trp 565 570 575cag cat gga gag gag gcc acg gtt aaa gcc gtg att gcg tgt atc ctc 1776 GlnHis Gly Glu Glu Ala Thr Val Lys Ala Val Ile Ala Cys Ile Leu 580 585 590tac cgg gca atg gcc cat gaa gct aag gag agt cac atg gtg gat gat 1824 TyrArg Ala Met Ala His Glu Ala Lys Glu Ser His Met Val Asp Asp 595 600 605gcc tca gaa gag ttg aag aat tac tca aaa cag ttt ggc cag ctg gct 1872 AlaSer Glu Glu Leu Lys Asn Tyr Ser Lys Gln Phe Gly Gln Leu Ala 610 615 620ctg gac ttg ttg gag aag gca ttc aag cag aat gag cgc atg gcc atg 1920 LeuAsp Leu Leu Glu Lys Ala Phe Lys Gln Asn Glu Arg Met Ala Met 625 630 635640 acg ctg ttg acg tat gaa ctc agg aac tgg agc aat tcg acc tgc ctg 1968Thr Leu Leu Thr Tyr Glu Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu 645 650655 aaa ctg gcc gtg tcg gga gga tta cga ccc ttt gtt tca cat act tgt 2016Lys Leu Ala Val Ser Gly Gly Leu Arg Pro Phe Val Ser His Thr Cys 660 665670 acc cag atg cta ctg aca gac atg tgg atg ggg agg ctg aaa atg agg 2064Thr Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Lys Met Arg 675 680685 aaa aac tct tgg tta aag att att ata agc att att tta cca ccc acc 2112Lys Asn Ser Trp Leu Lys Ile Ile Ile Ser Ile Ile Leu Pro Pro Thr 690 695700 att ttg aca ctg gaa ttt aaa agc aaa gct gag atg tca cat gtt ccc 2160Ile Leu Thr Leu Glu Phe Lys Ser Lys Ala Glu Met Ser His Val Pro 705 710715 720 cag tcc cag gac ttc caa ttt atg tgg tat tac agt gac cag aac gcc2208 Gln Ser Gln Asp Phe Gln Phe Met Trp Tyr Tyr Ser Asp Gln Asn Ala 725730 735 agc agt tcc aaa gaa agt gct tct gtg aaa gag tat gat ttg gaa agg2256 Ser Ser Ser Lys Glu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu Arg 740745 750 ggc cat gat gag aaa ctg gat gaa aat cag cat ttt ggt ttg gaa agt2304 Gly His Asp Glu Lys Leu Asp Glu Asn Gln His Phe Gly Leu Glu Ser 755760 765 ggg cac caa cac ctt ccg tgg acc agg aaa gtc tat gag ttc tac agt2352 Gly His Gln His Leu Pro Trp Thr Arg Lys Val Tyr Glu Phe Tyr Ser 770775 780 gct cca att gtc aag ttt tgg ttt tat acg atg gcg tat ttg gca ttc2400 Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala Phe 785790 795 800 ctc atg ctg ttc act tac acc gtg ttg gtg gag atg cag ccc cagccc 2448 Leu Met Leu Phe Thr Tyr Thr Val Leu Val Glu Met Gln Pro Gln Pro805 810 815 agc gtg cag gag tgg ctt gtt agc att tac atc ttc acc aat gctatt 2496 Ser Val Gln Glu Trp Leu Val Ser Ile Tyr Ile Phe Thr Asn Ala Ile820 825 830 gag gtg gtc agg gag atc tgt att tca gaa cct ggg aag ttt acccaa 2544 Glu Val Val Arg Glu Ile Cys Ile Ser Glu Pro Gly Lys Phe Thr Gln835 840 845 aag gtg aag gta tgg att agt gag tac tgg aac tta aca gaa actgtg 2592 Lys Val Lys Val Trp Ile Ser Glu Tyr Trp Asn Leu Thr Glu Thr Val850 855 860 gcc att ggc ctg ttt tca gct ggc ttc gtc ctt cga tgg ggt gaccct 2640 Ala Ile Gly Leu Phe Ser Ala Gly Phe Val Leu Arg Trp Gly Asp Pro865 870 875 880 cct ttt cac aca gcg gga aga ctg atc tac tgc ata gac atcata ttc 2688 Pro Phe His Thr Ala Gly Arg Leu Ile Tyr Cys Ile Asp Ile IlePhe 885 890 895 tgg ttc tca cgg ctc ctg gac ttc ttt gct gtg aat caa catgca ggt 2736 Trp Phe Ser Arg Leu Leu Asp Phe Phe Ala Val Asn Gln His AlaGly 900 905 910 cca tat gtg acc atg att gca aaa atg aca gca aac atg ttctat att 2784 Pro Tyr Val Thr Met Ile Ala Lys Met Thr Ala Asn Met Phe TyrIle 915 920 925 gtg atc atc atg gcc ata gtc ctg ctg agc ttt gga gtg gcacgc aag 2832 Val Ile Ile Met Ala Ile Val Leu Leu Ser Phe Gly Val Ala ArgLys 930 935 940 gcc atc ctt tcg cca aaa gag cca cca tct tgg agt cta gctcga gat 2880 Ala Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp Ser Leu Ala ArgAsp 945 950 955 960 att gta ttt gag cca tac tgg atg ata tac gga gaa gtctat gct gga 2928 Ile Val Phe Glu Pro Tyr Trp Met Ile Tyr Gly Glu Val TyrAla Gly 965 970 975 gaa ata gat gtt tgt tca agc cag cca tcc tgc cct cctggt tct ttt 2976 Glu Ile Asp Val Cys Ser Ser Gln Pro Ser Cys Pro Pro GlySer Phe 980 985 990 ctt act cca ttc ttg caa gct gtc tac ctc ttc gtg caatat atc atc 3024 Leu Thr Pro Phe Leu Gln Ala Val Tyr Leu Phe Val Gln TyrIle Ile 995 1000 1005 atg gtg aac ctg ttg att gct ttc ttc aac aac gtttac tta gat 3069 Met Val Asn Leu Leu Ile Ala Phe Phe Asn Asn Val Tyr LeuAsp 1010 1015 1020 atg gaa tcc att tca aat aac ctg tgg aaa tac aac cgctat cgc 3114 Met Glu Ser Ile Ser Asn Asn Leu Trp Lys Tyr Asn Arg Tyr Arg1025 1030 1035 tac atc atg acc tac cac gag aag ccc tgg ctg ccc cca cctctc 3159 Tyr Ile Met Thr Tyr His Glu Lys Pro Trp Leu Pro Pro Pro Leu1040 1045 1050 atc ctg ctg agc cac gtg ggc ctt ctc ctc cgc cgc ctg tgctgt 3204 Ile Leu Leu Ser His Val Gly Leu Leu Leu Arg Arg Leu Cys Cys1055 1060 1065 cat cga gct cct cac gac caa gaa gag ggt gac gtt gga ttaaaa 3249 His Arg Ala Pro His Asp Gln Glu Glu Gly Asp Val Gly Leu Lys1070 1075 1080 ctc tac ctc agt aag gag gat ctg aaa aaa ctt cat gat tttgag 3294 Leu Tyr Leu Ser Lys Glu Asp Leu Lys Lys Leu His Asp Phe Glu1085 1090 1095 gag cag tgc gtg gaa aaa tac ttc cat gag aag atg gaa gatgtg 3339 Glu Gln Cys Val Glu Lys Tyr Phe His Glu Lys Met Glu Asp Val1100 1105 1110 aat tgt agt tgt gag gaa cga atc cga gtg aca tca gaa agggtt 3384 Asn Cys Ser Cys Glu Glu Arg Ile Arg Val Thr Ser Glu Arg Val1115 1120 1125 aca gag atg tac ttc cag ctg aaa gaa atg aat gaa aag gtgtct 3429 Thr Glu Met Tyr Phe Gln Leu Lys Glu Met Asn Glu Lys Val Ser1130 1135 1140 ttt ata aag gac tcc tta ctg tct ttg gac agc cag gtg ggacac 3474 Phe Ile Lys Asp Ser Leu Leu Ser Leu Asp Ser Gln Val Gly His1145 1150 1155 ctg cag gat ctc tct gcc ctg act gtg gat acc ctg aaa gtcctt 3519 Leu Gln Asp Leu Ser Ala Leu Thr Val Asp Thr Leu Lys Val Leu1160 1165 1170 tct gct gtt gac act ttg caa gag gat gag gct ctc ctg gccaag 3564 Ser Ala Val Asp Thr Leu Gln Glu Asp Glu Ala Leu Leu Ala Lys1175 1180 1185 aga aag cat tct act tgc aaa aaa ctt ccc cac agc tgg agcaat 3609 Arg Lys His Ser Thr Cys Lys Lys Leu Pro His Ser Trp Ser Asn1190 1195 1200 gtc atc tgt gca gag gtt cta ggc agc atg gag atc gct ggagag 3654 Val Ile Cys Ala Glu Val Leu Gly Ser Met Glu Ile Ala Gly Glu1205 1210 1215 aag aaa tac cag tat tat agc atg ccc tct tct ttg ctg aggagc 3699 Lys Lys Tyr Gln Tyr Tyr Ser Met Pro Ser Ser Leu Leu Arg Ser1220 1225 1230 ctg gct gga ggc cgg cat ccc cca aga gtg cag agg ggg gcactt 3744 Leu Ala Gly Gly Arg His Pro Pro Arg Val Gln Arg Gly Ala Leu1235 1240 1245 ctt gag att aca aac agt aaa aga gag gct aca aat gta agaaat 3789 Leu Glu Ile Thr Asn Ser Lys Arg Glu Ala Thr Asn Val Arg Asn1250 1255 1260 gac cag gaa agg caa gaa aca caa agt agt ata gtg gtt tctggg 3834 Asp Gln Glu Arg Gln Glu Thr Gln Ser Ser Ile Val Val Ser Gly1265 1270 1275 gtg tct cct aac agg caa gca cac tca aag tat ggc cag tttctt 3879 Val Ser Pro Asn Arg Gln Ala His Ser Lys Tyr Gly Gln Phe Leu1280 1285 1290 ctg gtc ccc tct aat cta aag cga gtt cct ttt tca gca gaaact 3924 Leu Val Pro Ser Asn Leu Lys Arg Val Pro Phe Ser Ala Glu Thr1295 1300 1305 gtc ttg cct ctg tcc aga ccc tct gtg cca gat gtg ctg gcaact 3969 Val Leu Pro Leu Ser Arg Pro Ser Val Pro Asp Val Leu Ala Thr1310 1315 1320 gaa cag gac atc cag act gag gtt ctt gtt cat ctg act gggcag 4014 Glu Gln Asp Ile Gln Thr Glu Val Leu Val His Leu Thr Gly Gln1325 1330 1335 acc cca gtt gtc tct gac tgg gca tca gtg gat gaa ccc aaggaa 4059 Thr Pro Val Val Ser Asp Trp Ala Ser Val Asp Glu Pro Lys Glu1340 1345 1350 aag cac gag cct att gct cac tta ctg gat gga caa gac aaggca 4104 Lys His Glu Pro Ile Ala His Leu Leu Asp Gly Gln Asp Lys Ala1355 1360 1365 gag caa gtg cta ccc act ttg agt tgc aca cct gaa ccc atgaca 4149 Glu Gln Val Leu Pro Thr Leu Ser Cys Thr Pro Glu Pro Met Thr1370 1375 1380 atg agc tcc cct ctt tcc caa gcc aag atc atg caa act ggaggt 4194 Met Ser Ser Pro Leu Ser Gln Ala Lys Ile Met Gln Thr Gly Gly1385 1390 1395 gga tat gta aac tgg gca ttt tca gaa ggt gat gaa act ggtgtg 4239 Gly Tyr Val Asn Trp Ala Phe Ser Glu Gly Asp Glu Thr Gly Val1400 1405 1410 ttt agc atc aag aaa aag tgg caa acc tgc ttg ccc tcc acttgt 4284 Phe Ser Ile Lys Lys Lys Trp Gln Thr Cys Leu Pro Ser Thr Cys1415 1420 1425 gac agt gat tcc tct cgg agt gaa cag cac cag aag cag gcccag 4329 Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln Lys Gln Ala Gln1430 1435 1440 gac agc tcc cta tct gat aac tca aca aga tcg gcc cag agtagt 4374 Asp Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln Ser Ser1445 1450 1455 gaa tgc tca gag gtg gga cca tgg ctt cag cca aac aca tccttt 4419 Glu Cys Ser Glu Val Gly Pro Trp Leu Gln Pro Asn Thr Ser Phe1460 1465 1470 tgg atc aat cct ctc cgc aga tac agg ccc ttc gct agg agtcat 4464 Trp Ile Asn Pro Leu Arg Arg Tyr Arg Pro Phe Ala Arg Ser His1475 1480 1485 agt ttt aga ttc cat aag gag gag aaa ttg atg aag atc tgtaag 4509 Ser Phe Arg Phe His Lys Glu Glu Lys Leu Met Lys Ile Cys Lys1490 1495 1500 att aaa aat ctt tca ggc tct tca gaa ata ggg cag gga gcatgg 4554 Ile Lys Asn Leu Ser Gly Ser Ser Glu Ile Gly Gln Gly Ala Trp1505 1510 1515 gtc aaa gcg aaa atg cta acc aaa gac agg aga ctg tca aagaaa 4599 Val Lys Ala Lys Met Leu Thr Lys Asp Arg Arg Leu Ser Lys Lys1520 1525 1530 aag aag aat act caa gga ctc cag gtg cca atc ata aca gtcaat 4644 Lys Lys Asn Thr Gln Gly Leu Gln Val Pro Ile Ile Thr Val Asn1535 1540 1545 gcc tgc tct cag agt gac cag ttg aat cca gag cca gga gaaaac 4689 Ala Cys Ser Gln Ser Asp Gln Leu Asn Pro Glu Pro Gly Glu Asn1550 1555 1560 agc atc tct gaa gag gag tac agc aag aac tgg ttc aca gtgtcc 4734 Ser Ile Ser Glu Glu Glu Tyr Ser Lys Asn Trp Phe Thr Val Ser1565 1570 1575 aaa ttt agt cac aca ggt gta gaa cct tac ata cat cag aaaatg 4779 Lys Phe Ser His Thr Gly Val Glu Pro Tyr Ile His Gln Lys Met1580 1585 1590 aaa act aaa gaa att gga caa tgt gct ata caa atc agt gattac 4824 Lys Thr Lys Glu Ile Gly Gln Cys Ala Ile Gln Ile Ser Asp Tyr1595 1600 1605 cta aag cag tct caa gag gat ctc agc aaa aac tct ttg tggaat 4869 Leu Lys Gln Ser Gln Glu Asp Leu Ser Lys Asn Ser Leu Trp Asn1610 1615 1620 tcc agg agc acc aac ctc aat agg aac tcc ctg ctg aaa agttca 4914 Ser Arg Ser Thr Asn Leu Asn Arg Asn Ser Leu Leu Lys Ser Ser1625 1630 1635 att gga gtt gac aag atc tca gcc tcc tta aaa agc cct caagag 4959 Ile Gly Val Asp Lys Ile Ser Ala Ser Leu Lys Ser Pro Gln Glu1640 1645 1650 cct cac cat cat tat tca gcc att gaa agg aat aat tta atgagg 5004 Pro His His His Tyr Ser Ala Ile Glu Arg Asn Asn Leu Met Arg1655 1660 1665 ctt tct cag acc ata cca ttt aca cca gtc caa ctg ttt gcagga 5049 Leu Ser Gln Thr Ile Pro Phe Thr Pro Val Gln Leu Phe Ala Gly1670 1675 1680 gaa gaa ata act gtc tac agg ttg gag gag agt tcc cct ttaaac 5094 Glu Glu Ile Thr Val Tyr Arg Leu Glu Glu Ser Ser Pro Leu Asn1685 1690 1695 ctt gat aaa agc atg tcc tct tgg tct cag cgt ggg aga gcggca 5139 Leu Asp Lys Ser Met Ser Ser Trp Ser Gln Arg Gly Arg Ala Ala1700 1705 1710 atg atc cag gta ttg tcc cga gag gag atg gat ggg ggc ctccgt 5184 Met Ile Gln Val Leu Ser Arg Glu Glu Met Asp Gly Gly Leu Arg1715 1720 1725 aaa gct atg aga gtc gtc agc act tgg tct gag gat gac attctc 5229 Lys Ala Met Arg Val Val Ser Thr Trp Ser Glu Asp Asp Ile Leu1730 1735 1740 aag ccg gga caa gtt ttc att gtc aag tcc ttt ctt cct gaggtt 5274 Lys Pro Gly Gln Val Phe Ile Val Lys Ser Phe Leu Pro Glu Val1745 1750 1755 gtg cgg aca tgg cat aaa atc ttc cag gag agc act gtg cttcat 5319 Val Arg Thr Trp His Lys Ile Phe Gln Glu Ser Thr Val Leu His1760 1765 1770 ctt tgc ctc agg gaa att caa caa caa aga gct gct caa aaattg 5364 Leu Cys Leu Arg Glu Ile Gln Gln Gln Arg Ala Ala Gln Lys Leu1775 1780 1785 atc tat acc ttc aac caa gtg aaa cca caa acc ata ccc tacaca 5409 Ile Tyr Thr Phe Asn Gln Val Lys Pro Gln Thr Ile Pro Tyr Thr1790 1795 1800 cca agg ttc ctg gaa gtt ttc tta atc tac tgc cat tca gccaac 5454 Pro Arg Phe Leu Glu Val Phe Leu Ile Tyr Cys His Ser Ala Asn1805 1810 1815 cag tgg ttg acc att gag aag tat atg aca ggg gag ttc cggaag 5499 Gln Trp Leu Thr Ile Glu Lys Tyr Met Thr Gly Glu Phe Arg Lys1820 1825 1830 tat aac aac aac aat ggt gat gaa atc acc ccc acc aac accctg 5544 Tyr Asn Asn Asn Asn Gly Asp Glu Ile Thr Pro Thr Asn Thr Leu1835 1840 1845 gag gag ctg atg ttg gct ttc tct cac tgg acc tat gag tacact 5589 Glu Glu Leu Met Leu Ala Phe Ser His Trp Thr Tyr Glu Tyr Thr1850 1855 1860 cgg gga gag ctg ctg gtt tta gat ttg caa ggt gtt gga gaaaat 5634 Arg Gly Glu Leu Leu Val Leu Asp Leu Gln Gly Val Gly Glu Asn1865 1870 1875 ttg aca gat cca tct gtt ata aaa cct gaa gtc aaa caa tcaaga 5679 Leu Thr Asp Pro Ser Val Ile Lys Pro Glu Val Lys Gln Ser Arg1880 1885 1890 gga atg gtg ttt gga ccg gcc aat ttg ggg gaa gat gca attaga 5724 Gly Met Val Phe Gly Pro Ala Asn Leu Gly Glu Asp Ala Ile Arg1895 1900 1905 aac ttc att gca aaa cat cat tgt aac tcc tgc tgc cgg aagctc 5769 Asn Phe Ile Ala Lys His His Cys Asn Ser Cys Cys Arg Lys Leu1910 1915 1920 aaa ctc ccg gat tta aaa aga aat gac tat tcc cct gaa aggata 5814 Lys Leu Pro Asp Leu Lys Arg Asn Asp Tyr Ser Pro Glu Arg Ile1925 1930 1935 aat tcc acc ttt gga ctt gag ata aaa ata gaa tca gct gaggag 5859 Asn Ser Thr Phe Gly Leu Glu Ile Lys Ile Glu Ser Ala Glu Glu1940 1945 1950 cct cca gca agg gag acg ggt aga aat tcc cca gaa gat gatatg 5904 Pro Pro Ala Arg Glu Thr Gly Arg Asn Ser Pro Glu Asp Asp Met1955 1960 1965 caa cta taa 5913 Gln Leu 1970 4 1970 PRT Homo sapiens 4Met Ile Ile Leu Ser Lys Ser Gln Lys Ser Trp Ile Lys Gly Val Phe 1 5 1015 Asp Lys Arg Glu Cys Ser Thr Ile Ile Pro Ser Ser Lys Asn Pro His 20 2530 Arg Cys Thr Pro Val Cys Gln Val Cys Gln Asn Leu Ile Arg Cys Tyr 35 4045 Cys Gly Arg Leu Ile Gly Asp His Ala Gly Ile Asp Tyr Ser Trp Thr 50 5560 Ile Ser Ala Ala Lys Gly Lys Glu Ser Glu Gln Trp Ser Val Glu Lys 65 7075 80 His Thr Thr Lys Ser Pro Thr Asp Thr Phe Gly Thr Ile Asn Phe Gln 8590 95 Asp Gly Glu His Thr His His Ala Lys Tyr Ile Arg Thr Ser Tyr Asp100 105 110 Thr Lys Leu Asp His Leu Leu His Leu Met Leu Lys Glu Trp LysMet 115 120 125 Glu Leu Pro Lys Leu Val Ile Ser Val His Gly Gly Ile GlnAsn Phe 130 135 140 Thr Met Pro Ser Lys Phe Lys Glu Ile Phe Ser Gln GlyLeu Val Lys 145 150 155 160 Ala Ala Glu Thr Thr Gly Ala Trp Ile Ile ThrGlu Gly Ile Asn Thr 165 170 175 Gly Val Ser Lys His Val Gly Asp Ala LeuLys Ser His Ser Ser His 180 185 190 Ser Leu Arg Lys Ile Trp Thr Val GlyIle Pro Pro Trp Gly Val Ile 195 200 205 Glu Asn Gln Arg Asp Leu Ile GlyLys Asp Val Val Cys Leu Tyr Gln 210 215 220 Thr Leu Asp Asn Pro Leu SerLys Leu Thr Thr Leu Asn Ser Met His 225 230 235 240 Ser His Phe Ile LeuSer Asp Asp Gly Thr Val Gly Lys Tyr Gly Asn 245 250 255 Glu Met Lys LeuArg Arg Asn Leu Glu Lys Tyr Leu Ser Leu Gln Lys 260 265 270 Ile His CysArg Ser Arg Gln Gly Val Pro Val Val Gly Leu Val Val 275 280 285 Glu GlyGly Pro Asn Val Ile Leu Ser Val Trp Glu Thr Val Lys Asp 290 295 300 LysAsp Pro Val Val Val Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu 305 310 315320 Leu Ala Phe Thr His Lys His Leu Ala Asp Glu Gly Met Leu Arg Pro 325330 335 Gln Val Lys Glu Glu Ile Ile Cys Met Ile Gln Asn Thr Phe Asn Phe340 345 350 Ser Leu Lys Gln Ser Lys His Leu Phe Gln Ile Leu Met Glu CysMet 355 360 365 Val His Arg Asp Cys Ile Thr Ile Phe Asp Ala Asp Ser GluGlu Gln 370 375 380 Gln Asp Leu Asp Leu Ala Ile Leu Thr Ala Leu Leu LysGly Thr Asn 385 390 395 400 Leu Ser Ala Ser Glu Gln Leu Asn Leu Ala MetAla Trp Asp Arg Val 405 410 415 Asp Ile Ala Lys Lys His Ile Leu Ile TyrGlu Gln His Trp Lys Pro 420 425 430 Asp Ala Leu Glu Gln Ala Met Ser AspAla Leu Val Met Asp Arg Val 435 440 445 Asp Phe Val Lys Leu Leu Ile GluTyr Gly Val Asn Leu His Arg Phe 450 455 460 Leu Thr Ile Pro Arg Leu GluGlu Leu Tyr Asn Thr Lys Gln Gly Pro 465 470 475 480 Thr Asn Thr Leu LeuHis His Leu Val Gln Asp Val Lys Gln His Gln 485 490 495 Arg His Ser SerGly Asn Arg Asn Glu Ser Ala Glu Ser Thr Leu His 500 505 510 Ser Gln PheIle Arg Thr Ala Gln Pro Tyr Lys Phe Lys Glu Lys Ser 515 520 525 Ile ValLeu His Lys Ser Arg Lys Lys Ser Lys Glu Gln Asn Val Ser 530 535 540 AspAsp Pro Glu Ser Thr Gly Phe Leu Tyr Pro Tyr Asn Asp Leu Leu 545 550 555560 Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Met Phe Phe Trp 565570 575 Gln His Gly Glu Glu Ala Thr Val Lys Ala Val Ile Ala Cys Ile Leu580 585 590 Tyr Arg Ala Met Ala His Glu Ala Lys Glu Ser His Met Val AspAsp 595 600 605 Ala Ser Glu Glu Leu Lys Asn Tyr Ser Lys Gln Phe Gly GlnLeu Ala 610 615 620 Leu Asp Leu Leu Glu Lys Ala Phe Lys Gln Asn Glu ArgMet Ala Met 625 630 635 640 Thr Leu Leu Thr Tyr Glu Leu Arg Asn Trp SerAsn Ser Thr Cys Leu 645 650 655 Lys Leu Ala Val Ser Gly Gly Leu Arg ProPhe Val Ser His Thr Cys 660 665 670 Thr Gln Met Leu Leu Thr Asp Met TrpMet Gly Arg Leu Lys Met Arg 675 680 685 Lys Asn Ser Trp Leu Lys Ile IleIle Ser Ile Ile Leu Pro Pro Thr 690 695 700 Ile Leu Thr Leu Glu Phe LysSer Lys Ala Glu Met Ser His Val Pro 705 710 715 720 Gln Ser Gln Asp PheGln Phe Met Trp Tyr Tyr Ser Asp Gln Asn Ala 725 730 735 Ser Ser Ser LysGlu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu Arg 740 745 750 Gly His AspGlu Lys Leu Asp Glu Asn Gln His Phe Gly Leu Glu Ser 755 760 765 Gly HisGln His Leu Pro Trp Thr Arg Lys Val Tyr Glu Phe Tyr Ser 770 775 780 AlaPro Ile Val Lys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala Phe 785 790 795800 Leu Met Leu Phe Thr Tyr Thr Val Leu Val Glu Met Gln Pro Gln Pro 805810 815 Ser Val Gln Glu Trp Leu Val Ser Ile Tyr Ile Phe Thr Asn Ala Ile820 825 830 Glu Val Val Arg Glu Ile Cys Ile Ser Glu Pro Gly Lys Phe ThrGln 835 840 845 Lys Val Lys Val Trp Ile Ser Glu Tyr Trp Asn Leu Thr GluThr Val 850 855 860 Ala Ile Gly Leu Phe Ser Ala Gly Phe Val Leu Arg TrpGly Asp Pro 865 870 875 880 Pro Phe His Thr Ala Gly Arg Leu Ile Tyr CysIle Asp Ile Ile Phe 885 890 895 Trp Phe Ser Arg Leu Leu Asp Phe Phe AlaVal Asn Gln His Ala Gly 900 905 910 Pro Tyr Val Thr Met Ile Ala Lys MetThr Ala Asn Met Phe Tyr Ile 915 920 925 Val Ile Ile Met Ala Ile Val LeuLeu Ser Phe Gly Val Ala Arg Lys 930 935 940 Ala Ile Leu Ser Pro Lys GluPro Pro Ser Trp Ser Leu Ala Arg Asp 945 950 955 960 Ile Val Phe Glu ProTyr Trp Met Ile Tyr Gly Glu Val Tyr Ala Gly 965 970 975 Glu Ile Asp ValCys Ser Ser Gln Pro Ser Cys Pro Pro Gly Ser Phe 980 985 990 Leu Thr ProPhe Leu Gln Ala Val Tyr Leu Phe Val Gln Tyr Ile Ile 995 1000 1005 MetVal Asn Leu Leu Ile Ala Phe Phe Asn Asn Val Tyr Leu Asp 1010 1015 1020Met Glu Ser Ile Ser Asn Asn Leu Trp Lys Tyr Asn Arg Tyr Arg 1025 10301035 Tyr Ile Met Thr Tyr His Glu Lys Pro Trp Leu Pro Pro Pro Leu 10401045 1050 Ile Leu Leu Ser His Val Gly Leu Leu Leu Arg Arg Leu Cys Cys1055 1060 1065 His Arg Ala Pro His Asp Gln Glu Glu Gly Asp Val Gly LeuLys 1070 1075 1080 Leu Tyr Leu Ser Lys Glu Asp Leu Lys Lys Leu His AspPhe Glu 1085 1090 1095 Glu Gln Cys Val Glu Lys Tyr Phe His Glu Lys MetGlu Asp Val 1100 1105 1110 Asn Cys Ser Cys Glu Glu Arg Ile Arg Val ThrSer Glu Arg Val 1115 1120 1125 Thr Glu Met Tyr Phe Gln Leu Lys Glu MetAsn Glu Lys Val Ser 1130 1135 1140 Phe Ile Lys Asp Ser Leu Leu Ser LeuAsp Ser Gln Val Gly His 1145 1150 1155 Leu Gln Asp Leu Ser Ala Leu ThrVal Asp Thr Leu Lys Val Leu 1160 1165 1170 Ser Ala Val Asp Thr Leu GlnGlu Asp Glu Ala Leu Leu Ala Lys 1175 1180 1185 Arg Lys His Ser Thr CysLys Lys Leu Pro His Ser Trp Ser Asn 1190 1195 1200 Val Ile Cys Ala GluVal Leu Gly Ser Met Glu Ile Ala Gly Glu 1205 1210 1215 Lys Lys Tyr GlnTyr Tyr Ser Met Pro Ser Ser Leu Leu Arg Ser 1220 1225 1230 Leu Ala GlyGly Arg His Pro Pro Arg Val Gln Arg Gly Ala Leu 1235 1240 1245 Leu GluIle Thr Asn Ser Lys Arg Glu Ala Thr Asn Val Arg Asn 1250 1255 1260 AspGln Glu Arg Gln Glu Thr Gln Ser Ser Ile Val Val Ser Gly 1265 1270 1275Val Ser Pro Asn Arg Gln Ala His Ser Lys Tyr Gly Gln Phe Leu 1280 12851290 Leu Val Pro Ser Asn Leu Lys Arg Val Pro Phe Ser Ala Glu Thr 12951300 1305 Val Leu Pro Leu Ser Arg Pro Ser Val Pro Asp Val Leu Ala Thr1310 1315 1320 Glu Gln Asp Ile Gln Thr Glu Val Leu Val His Leu Thr GlyGln 1325 1330 1335 Thr Pro Val Val Ser Asp Trp Ala Ser Val Asp Glu ProLys Glu 1340 1345 1350 Lys His Glu Pro Ile Ala His Leu Leu Asp Gly GlnAsp Lys Ala 1355 1360 1365 Glu Gln Val Leu Pro Thr Leu Ser Cys Thr ProGlu Pro Met Thr 1370 1375 1380 Met Ser Ser Pro Leu Ser Gln Ala Lys IleMet Gln Thr Gly Gly 1385 1390 1395 Gly Tyr Val Asn Trp Ala Phe Ser GluGly Asp Glu Thr Gly Val 1400 1405 1410 Phe Ser Ile Lys Lys Lys Trp GlnThr Cys Leu Pro Ser Thr Cys 1415 1420 1425 Asp Ser Asp Ser Ser Arg SerGlu Gln His Gln Lys Gln Ala Gln 1430 1435 1440 Asp Ser Ser Leu Ser AspAsn Ser Thr Arg Ser Ala Gln Ser Ser 1445 1450 1455 Glu Cys Ser Glu ValGly Pro Trp Leu Gln Pro Asn Thr Ser Phe 1460 1465 1470 Trp Ile Asn ProLeu Arg Arg Tyr Arg Pro Phe Ala Arg Ser His 1475 1480 1485 Ser Phe ArgPhe His Lys Glu Glu Lys Leu Met Lys Ile Cys Lys 1490 1495 1500 Ile LysAsn Leu Ser Gly Ser Ser Glu Ile Gly Gln Gly Ala Trp 1505 1510 1515 ValLys Ala Lys Met Leu Thr Lys Asp Arg Arg Leu Ser Lys Lys 1520 1525 1530Lys Lys Asn Thr Gln Gly Leu Gln Val Pro Ile Ile Thr Val Asn 1535 15401545 Ala Cys Ser Gln Ser Asp Gln Leu Asn Pro Glu Pro Gly Glu Asn 15501555 1560 Ser Ile Ser Glu Glu Glu Tyr Ser Lys Asn Trp Phe Thr Val Ser1565 1570 1575 Lys Phe Ser His Thr Gly Val Glu Pro Tyr Ile His Gln LysMet 1580 1585 1590 Lys Thr Lys Glu Ile Gly Gln Cys Ala Ile Gln Ile SerAsp Tyr 1595 1600 1605 Leu Lys Gln Ser Gln Glu Asp Leu Ser Lys Asn SerLeu Trp Asn 1610 1615 1620 Ser Arg Ser Thr Asn Leu Asn Arg Asn Ser LeuLeu Lys Ser Ser 1625 1630 1635 Ile Gly Val Asp Lys Ile Ser Ala Ser LeuLys Ser Pro Gln Glu 1640 1645 1650 Pro His His His Tyr Ser Ala Ile GluArg Asn Asn Leu Met Arg 1655 1660 1665 Leu Ser Gln Thr Ile Pro Phe ThrPro Val Gln Leu Phe Ala Gly 1670 1675 1680 Glu Glu Ile Thr Val Tyr ArgLeu Glu Glu Ser Ser Pro Leu Asn 1685 1690 1695 Leu Asp Lys Ser Met SerSer Trp Ser Gln Arg Gly Arg Ala Ala 1700 1705 1710 Met Ile Gln Val LeuSer Arg Glu Glu Met Asp Gly Gly Leu Arg 1715 1720 1725 Lys Ala Met ArgVal Val Ser Thr Trp Ser Glu Asp Asp Ile Leu 1730 1735 1740 Lys Pro GlyGln Val Phe Ile Val Lys Ser Phe Leu Pro Glu Val 1745 1750 1755 Val ArgThr Trp His Lys Ile Phe Gln Glu Ser Thr Val Leu His 1760 1765 1770 LeuCys Leu Arg Glu Ile Gln Gln Gln Arg Ala Ala Gln Lys Leu 1775 1780 1785Ile Tyr Thr Phe Asn Gln Val Lys Pro Gln Thr Ile Pro Tyr Thr 1790 17951800 Pro Arg Phe Leu Glu Val Phe Leu Ile Tyr Cys His Ser Ala Asn 18051810 1815 Gln Trp Leu Thr Ile Glu Lys Tyr Met Thr Gly Glu Phe Arg Lys1820 1825 1830 Tyr Asn Asn Asn Asn Gly Asp Glu Ile Thr Pro Thr Asn ThrLeu 1835 1840 1845 Glu Glu Leu Met Leu Ala Phe Ser His Trp Thr Tyr GluTyr Thr 1850 1855 1860 Arg Gly Glu Leu Leu Val Leu Asp Leu Gln Gly ValGly Glu Asn 1865 1870 1875 Leu Thr Asp Pro Ser Val Ile Lys Pro Glu ValLys Gln Ser Arg 1880 1885 1890 Gly Met Val Phe Gly Pro Ala Asn Leu GlyGlu Asp Ala Ile Arg 1895 1900 1905 Asn Phe Ile Ala Lys His His Cys AsnSer Cys Cys Arg Lys Leu 1910 1915 1920 Lys Leu Pro Asp Leu Lys Arg AsnAsp Tyr Ser Pro Glu Arg Ile 1925 1930 1935 Asn Ser Thr Phe Gly Leu GluIle Lys Ile Glu Ser Ala Glu Glu 1940 1945 1950 Pro Pro Ala Arg Glu ThrGly Arg Asn Ser Pro Glu Asp Asp Met 1955 1960 1965 Gln Leu 1970 5 5820DNA Homo sapiens CDS (1)..(5817) 5 atg att atc cta tct aag tcc cag aaatcc tgg att aaa gga gta ttt 48 Met Ile Ile Leu Ser Lys Ser Gln Lys SerTrp Ile Lys Gly Val Phe 1 5 10 15 gac aag aga gaa tgt agc aca atc ataccc agc tca aaa aat cct cac 96 Asp Lys Arg Glu Cys Ser Thr Ile Ile ProSer Ser Lys Asn Pro His 20 25 30 aga tgt act cca gta tgc caa gtc tgc cagaat tta atc agg tgt tac 144 Arg Cys Thr Pro Val Cys Gln Val Cys Gln AsnLeu Ile Arg Cys Tyr 35 40 45 tgt ggc cga ctg att gga gac cat gct ggg atagat tat tcc tgg acc 192 Cys Gly Arg Leu Ile Gly Asp His Ala Gly Ile AspTyr Ser Trp Thr 50 55 60 atc tca gct gcc aag ggt aaa gaa agt gaa caa tggtct gtt gaa aag 240 Ile Ser Ala Ala Lys Gly Lys Glu Ser Glu Gln Trp SerVal Glu Lys 65 70 75 80 cac aca acg aaa agc cca aca gat act ttt ggc acgatt aat ttc caa 288 His Thr Thr Lys Ser Pro Thr Asp Thr Phe Gly Thr IleAsn Phe Gln 85 90 95 gat gga gag cac acc cat cat gcc aag tat att aga acttct tat gat 336 Asp Gly Glu His Thr His His Ala Lys Tyr Ile Arg Thr SerTyr Asp 100 105 110 aca aaa ctg gat cat ctg tta cat tta atg ttg aaa gagtgg aaa atg 384 Thr Lys Leu Asp His Leu Leu His Leu Met Leu Lys Glu TrpLys Met 115 120 125 gaa ctg ccc aag ctt gtg atc tca gtc cat ggg ggc atccag aac ttt 432 Glu Leu Pro Lys Leu Val Ile Ser Val His Gly Gly Ile GlnAsn Phe 130 135 140 act atg ccc tct aaa ttt aaa gag att ttc agc caa ggtttg gtt aaa 480 Thr Met Pro Ser Lys Phe Lys Glu Ile Phe Ser Gln Gly LeuVal Lys 145 150 155 160 gct gca gag aca aca gga gcg tgg ata ata act gaaggc atc aat aca 528 Ala Ala Glu Thr Thr Gly Ala Trp Ile Ile Thr Glu GlyIle Asn Thr 165 170 175 gga gtg tcc aag cat gtt ggg gat gcc ttg aaa tcccat tcc tct cat 576 Gly Val Ser Lys His Val Gly Asp Ala Leu Lys Ser HisSer Ser His 180 185 190 tcc ttg aga aaa atc tgg aca gtt gga atc cct ccttgg ggt gtc att 624 Ser Leu Arg Lys Ile Trp Thr Val Gly Ile Pro Pro TrpGly Val Ile 195 200 205 gag aac cag aga gac ctt att gga aaa gat gtg gtgtgc ctg tac cag 672 Glu Asn Gln Arg Asp Leu Ile Gly Lys Asp Val Val CysLeu Tyr Gln 210 215 220 act ctg gat aac ccc ctc agc aag ctc aca aca ctcaac agc atg cac 720 Thr Leu Asp Asn Pro Leu Ser Lys Leu Thr Thr Leu AsnSer Met His 225 230 235 240 tcg cac ttc atc ctg tct gat gat ggg acc gtgggc aag tat gga aat 768 Ser His Phe Ile Leu Ser Asp Asp Gly Thr Val GlyLys Tyr Gly Asn 245 250 255 gaa atg aag ctc aga agg aac ctg gag aag tacctc tct ctg cag aaa 816 Glu Met Lys Leu Arg Arg Asn Leu Glu Lys Tyr LeuSer Leu Gln Lys 260 265 270 ata cac tgc cgc tca aga caa ggc gtg ccg gtcgtg ggg ctg gtg gtg 864 Ile His Cys Arg Ser Arg Gln Gly Val Pro Val ValGly Leu Val Val 275 280 285 gaa ggc ggt ccc aac gtc atc ctg tca gtg tgggag act gtc aag gac 912 Glu Gly Gly Pro Asn Val Ile Leu Ser Val Trp GluThr Val Lys Asp 290 295 300 aag gac cca gtg gtg gtg tgt gag ggc aca ggtagg gcg gct gac ctc 960 Lys Asp Pro Val Val Val Cys Glu Gly Thr Gly ArgAla Ala Asp Leu 305 310 315 320 ctg gcc ttc aca cac aaa cac ctg gca gatgaa ggg atg ctg cga cct 1008 Leu Ala Phe Thr His Lys His Leu Ala Asp GluGly Met Leu Arg Pro 325 330 335 cag gtg aaa gag gag atc atc tgc atg attcag aac act ttc aac ttt 1056 Gln Val Lys Glu Glu Ile Ile Cys Met Ile GlnAsn Thr Phe Asn Phe 340 345 350 agt ctt aaa cag tcc aag cac ctt ttc caaatt cta atg gag tgt atg 1104 Ser Leu Lys Gln Ser Lys His Leu Phe Gln IleLeu Met Glu Cys Met 355 360 365 gtt cac agg gat tgt att acc ata ttt gatgct gac tct gaa gag cag 1152 Val His Arg Asp Cys Ile Thr Ile Phe Asp AlaAsp Ser Glu Glu Gln 370 375 380 caa gac ctg gac tta gca atc cta aca gctttg ctg aag ggc aca aat 1200 Gln Asp Leu Asp Leu Ala Ile Leu Thr Ala LeuLeu Lys Gly Thr Asn 385 390 395 400 tta tca gcg tca gag caa tta aat ctggca atg gct tgg gac agg gtg 1248 Leu Ser Ala Ser Glu Gln Leu Asn Leu AlaMet Ala Trp Asp Arg Val 405 410 415 gac att gcc aag aaa cat atc cta atttat gaa caa cac tgg aag cct 1296 Asp Ile Ala Lys Lys His Ile Leu Ile TyrGlu Gln His Trp Lys Pro 420 425 430 gat gcc ctg gaa caa gca atg tca gatgct tta gtg atg gat cgg gtg 1344 Asp Ala Leu Glu Gln Ala Met Ser Asp AlaLeu Val Met Asp Arg Val 435 440 445 gat ttt gtg aag ctc tta ata gaa tatgga gtg aac ctc cat cgc ttt 1392 Asp Phe Val Lys Leu Leu Ile Glu Tyr GlyVal Asn Leu His Arg Phe 450 455 460 ctt acc atc cct cga ctg gaa gag ctctac aat aca aaa caa gga cct 1440 Leu Thr Ile Pro Arg Leu Glu Glu Leu TyrAsn Thr Lys Gln Gly Pro 465 470 475 480 act aat aca ctc ttg cat cat ctcgtc caa gat gtg aaa cag gaa aag 1488 Thr Asn Thr Leu Leu His His Leu ValGln Asp Val Lys Gln Glu Lys 485 490 495 tct ata gtc ctt cat aaa tca aggaag aag tca aaa gaa caa aat gta 1536 Ser Ile Val Leu His Lys Ser Arg LysLys Ser Lys Glu Gln Asn Val 500 505 510 tca gat gac cct gag tct act ggcttt ctt tac cct tac aat gac ctg 1584 Ser Asp Asp Pro Glu Ser Thr Gly PheLeu Tyr Pro Tyr Asn Asp Leu 515 520 525 ctg gtt tgg gct gtg ctg atg aaaagg cag aag atg gct atg ttc ttc 1632 Leu Val Trp Ala Val Leu Met Lys ArgGln Lys Met Ala Met Phe Phe 530 535 540 tgg cag cat gga gag gag gcc acggtt aaa gcc gtg att gcg tgt atc 1680 Trp Gln His Gly Glu Glu Ala Thr ValLys Ala Val Ile Ala Cys Ile 545 550 555 560 ctc tac cgg gca atg gcc catgaa gct aag gag agt cac atg gtg gat 1728 Leu Tyr Arg Ala Met Ala His GluAla Lys Glu Ser His Met Val Asp 565 570 575 gat gcc tca gaa gag ttg aagaat tac tca aaa cag ttt ggc cag ctg 1776 Asp Ala Ser Glu Glu Leu Lys AsnTyr Ser Lys Gln Phe Gly Gln Leu 580 585 590 gct ctg gac ttg ttg gag aaggca ttc aag cag aat gag cgc atg gcc 1824 Ala Leu Asp Leu Leu Glu Lys AlaPhe Lys Gln Asn Glu Arg Met Ala 595 600 605 atg acg ctg ttg acg tat gaactc agg aac tgg agc aat tcg acc tgc 1872 Met Thr Leu Leu Thr Tyr Glu LeuArg Asn Trp Ser Asn Ser Thr Cys 610 615 620 ctg aaa ctg gcc gtg tcg ggagga tta cga ccc ttt gtt tca cat act 1920 Leu Lys Leu Ala Val Ser Gly GlyLeu Arg Pro Phe Val Ser His Thr 625 630 635 640 tgt acc cag atg cta ctgaca gac atg tgg atg ggg agg ctg aaa atg 1968 Cys Thr Gln Met Leu Leu ThrAsp Met Trp Met Gly Arg Leu Lys Met 645 650 655 agg aaa aac tct tgg ttaaag att att ata agc att att tta cca ccc 2016 Arg Lys Asn Ser Trp Leu LysIle Ile Ile Ser Ile Ile Leu Pro Pro 660 665 670 acc att ttg aca ctg gaattt aaa agc aaa gct gag atg tca cat gtt 2064 Thr Ile Leu Thr Leu Glu PheLys Ser Lys Ala Glu Met Ser His Val 675 680 685 ccc cag tcc cag gac ttccaa ttt atg tgg tat tac agt gac cag aac 2112 Pro Gln Ser Gln Asp Phe GlnPhe Met Trp Tyr Tyr Ser Asp Gln Asn 690 695 700 gcc agc agt tcc aaa gaaagt gct tct gtg aaa gag tat gat ttg gaa 2160 Ala Ser Ser Ser Lys Glu SerAla Ser Val Lys Glu Tyr Asp Leu Glu 705 710 715 720 agg ggc cat gat gagaaa ctg gat gaa aat cag cat ttt ggt ttg gaa 2208 Arg Gly His Asp Glu LysLeu Asp Glu Asn Gln His Phe Gly Leu Glu 725 730 735 agt ggg cac caa cacctt ccg tgg acc agg aaa gtc tat gag ttc tac 2256 Ser Gly His Gln His LeuPro Trp Thr Arg Lys Val Tyr Glu Phe Tyr 740 745 750 agt gct cca att gtcaag ttt tgg ttt tat acg atg gcg tat ttg gca 2304 Ser Ala Pro Ile Val LysPhe Trp Phe Tyr Thr Met Ala Tyr Leu Ala 755 760 765 ttc ctc atg ctg ttcact tac acc gtg ttg gtg gag atg cag ccc cag 2352 Phe Leu Met Leu Phe ThrTyr Thr Val Leu Val Glu Met Gln Pro Gln 770 775 780 ccc agc gtg cag gagtgg ctt gtt agc att tac atc ttc acc aat gct 2400 Pro Ser Val Gln Glu TrpLeu Val Ser Ile Tyr Ile Phe Thr Asn Ala 785 790 795 800 att gag gtg gtcagg gag atc tgt att tca gaa cct ggg aag ttt acc 2448 Ile Glu Val Val ArgGlu Ile Cys Ile Ser Glu Pro Gly Lys Phe Thr 805 810 815 caa aag gtg aaggta tgg att agt gag tac tgg aac tta aca gaa act 2496 Gln Lys Val Lys ValTrp Ile Ser Glu Tyr Trp Asn Leu Thr Glu Thr 820 825 830 gtg gcc att ggcctg ttt tca gct ggc ttc gtc ctt cga tgg ggt gac 2544 Val Ala Ile Gly LeuPhe Ser Ala Gly Phe Val Leu Arg Trp Gly Asp 835 840 845 cct cct ttt cacaca gcg gga aga ctg atc tac tgc ata gac atc ata 2592 Pro Pro Phe His ThrAla Gly Arg Leu Ile Tyr Cys Ile Asp Ile Ile 850 855 860 ttc tgg ttc tcacgg ctc ctg gac ttc ttt gct gtg aat caa cat gca 2640 Phe Trp Phe Ser ArgLeu Leu Asp Phe Phe Ala Val Asn Gln His Ala 865 870 875 880 ggt cca tatgtg acc atg att gca aaa atg aca gca aac atg ttc tat 2688 Gly Pro Tyr ValThr Met Ile Ala Lys Met Thr Ala Asn Met Phe Tyr 885 890 895 att gtg atcatc atg gcc ata gtc ctg ctg agc ttt gga gtg gca cgc 2736 Ile Val Ile IleMet Ala Ile Val Leu Leu Ser Phe Gly Val Ala Arg 900 905 910 aag gcc atcctt tcg cca aaa gag cca cca tct tgg agt cta gct cga 2784 Lys Ala Ile LeuSer Pro Lys Glu Pro Pro Ser Trp Ser Leu Ala Arg 915 920 925 gat att gtattt gag cca tac tgg atg ata tac gga gaa gtc tat gct 2832 Asp Ile Val PheGlu Pro Tyr Trp Met Ile Tyr Gly Glu Val Tyr Ala 930 935 940 gga gaa atagat gtt tgt tca agc cag cca tcc tgc cct cct ggt tct 2880 Gly Glu Ile AspVal Cys Ser Ser Gln Pro Ser Cys Pro Pro Gly Ser 945 950 955 960 ttt cttact cca ttc ttg caa gct gtc tac ctc ttc gtg caa tat atc 2928 Phe Leu ThrPro Phe Leu Gln Ala Val Tyr Leu Phe Val Gln Tyr Ile 965 970 975 atc atggtg aac ctg ttg att gct ttc ttc aac aac gtt tac tta gat 2976 Ile Met ValAsn Leu Leu Ile Ala Phe Phe Asn Asn Val Tyr Leu Asp 980 985 990 atg gaatcc att tca aat aac ctg tgg aaa tac aac cgc tat cgc tac 3024 Met Glu SerIle Ser Asn Asn Leu Trp Lys Tyr Asn Arg Tyr Arg Tyr 995 1000 1005 atcatg acc tac cac gag aag ccc tgg ctg ccc cca cct ctc atc 3069 Ile Met ThrTyr His Glu Lys Pro Trp Leu Pro Pro Pro Leu Ile 1010 1015 1020 ctg ctgagc cac gtg ggc ctt ctc ctc cgc cgc ctg tgc tgt cat 3114 Leu Leu Ser HisVal Gly Leu Leu Leu Arg Arg Leu Cys Cys His 1025 1030 1035 cga gct cctcac gac caa gaa gag ggt gac gtt gga tta aaa ctc 3159 Arg Ala Pro His AspGln Glu Glu Gly Asp Val Gly Leu Lys Leu 1040 1045 1050 tac ctc agt aaggag gat ctg aaa aaa ctt cat gat ttt gag gag 3204 Tyr Leu Ser Lys Glu AspLeu Lys Lys Leu His Asp Phe Glu Glu 1055 1060 1065 cag tgc gtg gaa aaatac ttc cat gag aag atg gaa gat gtg aat 3249 Gln Cys Val Glu Lys Tyr PheHis Glu Lys Met Glu Asp Val Asn 1070 1075 1080 tgt agt tgt gag gaa cgaatc cga gtg aca tca gaa agg gtt aca 3294 Cys Ser Cys Glu Glu Arg Ile ArgVal Thr Ser Glu Arg Val Thr 1085 1090 1095 gag atg tac ttc cag ctg aaagaa atg aat gaa aag gtg tct ttt 3339 Glu Met Tyr Phe Gln Leu Lys Glu MetAsn Glu Lys Val Ser Phe 1100 1105 1110 ata aag gac tcc tta ctg tct ttggac agc cag gtg gga cac ctg 3384 Ile Lys Asp Ser Leu Leu Ser Leu Asp SerGln Val Gly His Leu 1115 1120 1125 cag gat ctc tct gcc ctg act gtg gatacc ctg aaa gtc ctt tct 3429 Gln Asp Leu Ser Ala Leu Thr Val Asp Thr LeuLys Val Leu Ser 1130 1135 1140 gct gtt gac act ttg caa gag gat gag gctctc ctg gcc aag aga 3474 Ala Val Asp Thr Leu Gln Glu Asp Glu Ala Leu LeuAla Lys Arg 1145 1150 1155 aag cat tct act tgc aaa aaa ctt ccc cac agctgg agc aat gtc 3519 Lys His Ser Thr Cys Lys Lys Leu Pro His Ser Trp SerAsn Val 1160 1165 1170 atc tgt gca gag gtt cta ggc agc atg gag atc gctgga gag aag 3564 Ile Cys Ala Glu Val Leu Gly Ser Met Glu Ile Ala Gly GluLys 1175 1180 1185 aaa tac cag tat tat agc atg ccc tct tct ttg ctg aggagc ctg 3609 Lys Tyr Gln Tyr Tyr Ser Met Pro Ser Ser Leu Leu Arg Ser Leu1190 1195 1200 gct gga ggc cgg cat ccc cca aga gtg cag agg ggg gca cttctt 3654 Ala Gly Gly Arg His Pro Pro Arg Val Gln Arg Gly Ala Leu Leu1205 1210 1215 gag att aca aac agt aaa aga gag gct aca aat gta aga aatgac 3699 Glu Ile Thr Asn Ser Lys Arg Glu Ala Thr Asn Val Arg Asn Asp1220 1225 1230 cag gaa agg caa gaa aca caa agt agt ata gtg gtt tct ggggtg 3744 Gln Glu Arg Gln Glu Thr Gln Ser Ser Ile Val Val Ser Gly Val1235 1240 1245 tct cct aac agg caa gca cac tca aag tat ggc cag ttt cttctg 3789 Ser Pro Asn Arg Gln Ala His Ser Lys Tyr Gly Gln Phe Leu Leu1250 1255 1260 gtc ccc tct aat cta aag cga gtt cct ttt tca gca gaa actgtc 3834 Val Pro Ser Asn Leu Lys Arg Val Pro Phe Ser Ala Glu Thr Val1265 1270 1275 ttg cct ctg tcc aga ccc tct gtg cca gat gtg ctg gca actgaa 3879 Leu Pro Leu Ser Arg Pro Ser Val Pro Asp Val Leu Ala Thr Glu1280 1285 1290 cag gac atc cag act gag gtt ctt gtt cat ctg act ggg cagacc 3924 Gln Asp Ile Gln Thr Glu Val Leu Val His Leu Thr Gly Gln Thr1295 1300 1305 cca gtt gtc tct gac tgg gca tca gtg gat gaa ccc aag gaaaag 3969 Pro Val Val Ser Asp Trp Ala Ser Val Asp Glu Pro Lys Glu Lys1310 1315 1320 cac gag cct att gct cac tta ctg gat gga caa gac aag gcagag 4014 His Glu Pro Ile Ala His Leu Leu Asp Gly Gln Asp Lys Ala Glu1325 1330 1335 caa gtg cta ccc act ttg agt tgc aca cct gaa ccc atg acaatg 4059 Gln Val Leu Pro Thr Leu Ser Cys Thr Pro Glu Pro Met Thr Met1340 1345 1350 agc tcc cct ctt tcc caa gcc aag atc atg caa act gga ggtgga 4104 Ser Ser Pro Leu Ser Gln Ala Lys Ile Met Gln Thr Gly Gly Gly1355 1360 1365 tat gta aac tgg gca ttt tca gaa ggt gat gaa act ggt gtgttt 4149 Tyr Val Asn Trp Ala Phe Ser Glu Gly Asp Glu Thr Gly Val Phe1370 1375 1380 agc atc aag aaa aag tgg caa acc tgc ttg ccc tcc act tgtgac 4194 Ser Ile Lys Lys Lys Trp Gln Thr Cys Leu Pro Ser Thr Cys Asp1385 1390 1395 agt gat tcc tct cgg agt gaa cag cac cag aag cag gcc caggac 4239 Ser Asp Ser Ser Arg Ser Glu Gln His Gln Lys Gln Ala Gln Asp1400 1405 1410 agc tcc cta tct gat aac tca aca aga tcg gcc cag agt agtgaa 4284 Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln Ser Ser Glu1415 1420 1425 tgc tca gag gtg gga cca tgg ctt cag cca aac aca tcc ttttgg 4329 Cys Ser Glu Val Gly Pro Trp Leu Gln Pro Asn Thr Ser Phe Trp1430 1435 1440 atc aat cct ctc cgc aga tac agg ccc ttc gct agg agt catagt 4374 Ile Asn Pro Leu Arg Arg Tyr Arg Pro Phe Ala Arg Ser His Ser1445 1450 1455 ttt aga ttc cat aag gag gag aaa ttg atg aag atc tgt aagatt 4419 Phe Arg Phe His Lys Glu Glu Lys Leu Met Lys Ile Cys Lys Ile1460 1465 1470 aaa aat ctt tca ggc tct tca gaa ata ggg cag gga gca tgggtc 4464 Lys Asn Leu Ser Gly Ser Ser Glu Ile Gly Gln Gly Ala Trp Val1475 1480 1485 aaa gcg aaa atg cta acc aaa gac agg aga ctg tca aag aaaaag 4509 Lys Ala Lys Met Leu Thr Lys Asp Arg Arg Leu Ser Lys Lys Lys1490 1495 1500 aag aat act caa gga ctc cag gtg cca atc ata aca gtc aatgcc 4554 Lys Asn Thr Gln Gly Leu Gln Val Pro Ile Ile Thr Val Asn Ala1505 1510 1515 tgc tct cag agt gac cag ttg aat cca gag cca gga gaa aacagc 4599 Cys Ser Gln Ser Asp Gln Leu Asn Pro Glu Pro Gly Glu Asn Ser1520 1525 1530 atc tct gaa gag gag tac agc aag aac tgg ttc aca gtg tccaaa 4644 Ile Ser Glu Glu Glu Tyr Ser Lys Asn Trp Phe Thr Val Ser Lys1535 1540 1545 ttt agt cac aca ggt gta gaa cct tac ata cat cag aaa atgaaa 4689 Phe Ser His Thr Gly Val Glu Pro Tyr Ile His Gln Lys Met Lys1550 1555 1560 act aaa gaa att gga caa tgt gct ata caa atc agt gat taccta 4734 Thr Lys Glu Ile Gly Gln Cys Ala Ile Gln Ile Ser Asp Tyr Leu1565 1570 1575 aag cag tct caa gag gat ctc agc aaa aac tct ttg tgg aattcc 4779 Lys Gln Ser Gln Glu Asp Leu Ser Lys Asn Ser Leu Trp Asn Ser1580 1585 1590 agg agc acc aac ctc aat agg aac tcc ctg ctg aaa agt tcaatt 4824 Arg Ser Thr Asn Leu Asn Arg Asn Ser Leu Leu Lys Ser Ser Ile1595 1600 1605 gga gtt gac aag atc tca gcc tcc tta aaa agc cct caa gagcct 4869 Gly Val Asp Lys Ile Ser Ala Ser Leu Lys Ser Pro Gln Glu Pro1610 1615 1620 cac cat cat tat tca gcc att gaa agg aat aat tta atg aggctt 4914 His His His Tyr Ser Ala Ile Glu Arg Asn Asn Leu Met Arg Leu1625 1630 1635 tct cag acc ata cca ttt aca cca gtc caa ctg ttt gca ggagaa 4959 Ser Gln Thr Ile Pro Phe Thr Pro Val Gln Leu Phe Ala Gly Glu1640 1645 1650 gaa ata act gtc tac agg ttg gag gag agt tcc cct tta aacctt 5004 Glu Ile Thr Val Tyr Arg Leu Glu Glu Ser Ser Pro Leu Asn Leu1655 1660 1665 gat aaa agc atg tcc tct tgg tct cag cgt ggg aga gcg gcaatg 5049 Asp Lys Ser Met Ser Ser Trp Ser Gln Arg Gly Arg Ala Ala Met1670 1675 1680 atc cag gta ttg tcc cga gag gag atg gat ggg ggc ctc cgtaaa 5094 Ile Gln Val Leu Ser Arg Glu Glu Met Asp Gly Gly Leu Arg Lys1685 1690 1695 gct atg aga gtc gtc agc act tgg tct gag gat gac att ctcaag 5139 Ala Met Arg Val Val Ser Thr Trp Ser Glu Asp Asp Ile Leu Lys1700 1705 1710 ccg gga caa gtt ttc att gtc aag tcc ttt ctt cct gag gttgtg 5184 Pro Gly Gln Val Phe Ile Val Lys Ser Phe Leu Pro Glu Val Val1715 1720 1725 cgg aca tgg cat aaa atc ttc cag gag agc act gtg ctt catctt 5229 Arg Thr Trp His Lys Ile Phe Gln Glu Ser Thr Val Leu His Leu1730 1735 1740 tgc ctc agg gaa att caa caa caa aga gct gct caa aaa ttgatc 5274 Cys Leu Arg Glu Ile Gln Gln Gln Arg Ala Ala Gln Lys Leu Ile1745 1750 1755 tat acc ttc aac caa gtg aaa cca caa acc ata ccc tac acacca 5319 Tyr Thr Phe Asn Gln Val Lys Pro Gln Thr Ile Pro Tyr Thr Pro1760 1765 1770 agg ttc ctg gaa gtt ttc tta atc tac tgc cat tca gcc aaccag 5364 Arg Phe Leu Glu Val Phe Leu Ile Tyr Cys His Ser Ala Asn Gln1775 1780 1785 tgg ttg acc att gag aag tat atg aca ggg gag ttc cgg aagtat 5409 Trp Leu Thr Ile Glu Lys Tyr Met Thr Gly Glu Phe Arg Lys Tyr1790 1795 1800 aac aac aac aat ggt gat gaa atc acc ccc acc aac acc ctggag 5454 Asn Asn Asn Asn Gly Asp Glu Ile Thr Pro Thr Asn Thr Leu Glu1805 1810 1815 gag ctg atg ttg gct ttc tct cac tgg acc tat gag tac actcgg 5499 Glu Leu Met Leu Ala Phe Ser His Trp Thr Tyr Glu Tyr Thr Arg1820 1825 1830 gga gag ctg ctg gtt tta gat ttg caa ggt gtt gga gaa aatttg 5544 Gly Glu Leu Leu Val Leu Asp Leu Gln Gly Val Gly Glu Asn Leu1835 1840 1845 aca gat cca tct gtt ata aaa cct gaa gtc aaa caa tca agagga 5589 Thr Asp Pro Ser Val Ile Lys Pro Glu Val Lys Gln Ser Arg Gly1850 1855 1860 atg gtg ttt gga ccg gcc aat ttg ggg gaa gat gca att agaaac 5634 Met Val Phe Gly Pro Ala Asn Leu Gly Glu Asp Ala Ile Arg Asn1865 1870 1875 ttc att gca aaa cat cat tgt aac tcc tgc tgc cgg aag ctcaaa 5679 Phe Ile Ala Lys His His Cys Asn Ser Cys Cys Arg Lys Leu Lys1880 1885 1890 ctc ccg gat tta aaa aga aat gac tat tcc cct gaa agg ataaat 5724 Leu Pro Asp Leu Lys Arg Asn Asp Tyr Ser Pro Glu Arg Ile Asn1895 1900 1905 tcc acc ttt gga ctt gag ata aaa ata gaa tca gct gag gagcct 5769 Ser Thr Phe Gly Leu Glu Ile Lys Ile Glu Ser Ala Glu Glu Pro1910 1915 1920 cca gca agg gag acg ggt aga aat tcc cca gaa gat gat atgcaa 5814 Pro Ala Arg Glu Thr Gly Arg Asn Ser Pro Glu Asp Asp Met Gln1925 1930 1935 cta taa 5820 Leu 6 1939 PRT Homo sapiens 6 Met Ile IleLeu Ser Lys Ser Gln Lys Ser Trp Ile Lys Gly Val Phe 1 5 10 15 Asp LysArg Glu Cys Ser Thr Ile Ile Pro Ser Ser Lys Asn Pro His 20 25 30 Arg CysThr Pro Val Cys Gln Val Cys Gln Asn Leu Ile Arg Cys Tyr 35 40 45 Cys GlyArg Leu Ile Gly Asp His Ala Gly Ile Asp Tyr Ser Trp Thr 50 55 60 Ile SerAla Ala Lys Gly Lys Glu Ser Glu Gln Trp Ser Val Glu Lys 65 70 75 80 HisThr Thr Lys Ser Pro Thr Asp Thr Phe Gly Thr Ile Asn Phe Gln 85 90 95 AspGly Glu His Thr His His Ala Lys Tyr Ile Arg Thr Ser Tyr Asp 100 105 110Thr Lys Leu Asp His Leu Leu His Leu Met Leu Lys Glu Trp Lys Met 115 120125 Glu Leu Pro Lys Leu Val Ile Ser Val His Gly Gly Ile Gln Asn Phe 130135 140 Thr Met Pro Ser Lys Phe Lys Glu Ile Phe Ser Gln Gly Leu Val Lys145 150 155 160 Ala Ala Glu Thr Thr Gly Ala Trp Ile Ile Thr Glu Gly IleAsn Thr 165 170 175 Gly Val Ser Lys His Val Gly Asp Ala Leu Lys Ser HisSer Ser His 180 185 190 Ser Leu Arg Lys Ile Trp Thr Val Gly Ile Pro ProTrp Gly Val Ile 195 200 205 Glu Asn Gln Arg Asp Leu Ile Gly Lys Asp ValVal Cys Leu Tyr Gln 210 215 220 Thr Leu Asp Asn Pro Leu Ser Lys Leu ThrThr Leu Asn Ser Met His 225 230 235 240 Ser His Phe Ile Leu Ser Asp AspGly Thr Val Gly Lys Tyr Gly Asn 245 250 255 Glu Met Lys Leu Arg Arg AsnLeu Glu Lys Tyr Leu Ser Leu Gln Lys 260 265 270 Ile His Cys Arg Ser ArgGln Gly Val Pro Val Val Gly Leu Val Val 275 280 285 Glu Gly Gly Pro AsnVal Ile Leu Ser Val Trp Glu Thr Val Lys Asp 290 295 300 Lys Asp Pro ValVal Val Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu 305 310 315 320 Leu AlaPhe Thr His Lys His Leu Ala Asp Glu Gly Met Leu Arg Pro 325 330 335 GlnVal Lys Glu Glu Ile Ile Cys Met Ile Gln Asn Thr Phe Asn Phe 340 345 350Ser Leu Lys Gln Ser Lys His Leu Phe Gln Ile Leu Met Glu Cys Met 355 360365 Val His Arg Asp Cys Ile Thr Ile Phe Asp Ala Asp Ser Glu Glu Gln 370375 380 Gln Asp Leu Asp Leu Ala Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn385 390 395 400 Leu Ser Ala Ser Glu Gln Leu Asn Leu Ala Met Ala Trp AspArg Val 405 410 415 Asp Ile Ala Lys Lys His Ile Leu Ile Tyr Glu Gln HisTrp Lys Pro 420 425 430 Asp Ala Leu Glu Gln Ala Met Ser Asp Ala Leu ValMet Asp Arg Val 435 440 445 Asp Phe Val Lys Leu Leu Ile Glu Tyr Gly ValAsn Leu His Arg Phe 450 455 460 Leu Thr Ile Pro Arg Leu Glu Glu Leu TyrAsn Thr Lys Gln Gly Pro 465 470 475 480 Thr Asn Thr Leu Leu His His LeuVal Gln Asp Val Lys Gln Glu Lys 485 490 495 Ser Ile Val Leu His Lys SerArg Lys Lys Ser Lys Glu Gln Asn Val 500 505 510 Ser Asp Asp Pro Glu SerThr Gly Phe Leu Tyr Pro Tyr Asn Asp Leu 515 520 525 Leu Val Trp Ala ValLeu Met Lys Arg Gln Lys Met Ala Met Phe Phe 530 535 540 Trp Gln His GlyGlu Glu Ala Thr Val Lys Ala Val Ile Ala Cys Ile 545 550 555 560 Leu TyrArg Ala Met Ala His Glu Ala Lys Glu Ser His Met Val Asp 565 570 575 AspAla Ser Glu Glu Leu Lys Asn Tyr Ser Lys Gln Phe Gly Gln Leu 580 585 590Ala Leu Asp Leu Leu Glu Lys Ala Phe Lys Gln Asn Glu Arg Met Ala 595 600605 Met Thr Leu Leu Thr Tyr Glu Leu Arg Asn Trp Ser Asn Ser Thr Cys 610615 620 Leu Lys Leu Ala Val Ser Gly Gly Leu Arg Pro Phe Val Ser His Thr625 630 635 640 Cys Thr Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg LeuLys Met 645 650 655 Arg Lys Asn Ser Trp Leu Lys Ile Ile Ile Ser Ile IleLeu Pro Pro 660 665 670 Thr Ile Leu Thr Leu Glu Phe Lys Ser Lys Ala GluMet Ser His Val 675 680 685 Pro Gln Ser Gln Asp Phe Gln Phe Met Trp TyrTyr Ser Asp Gln Asn 690 695 700 Ala Ser Ser Ser Lys Glu Ser Ala Ser ValLys Glu Tyr Asp Leu Glu 705 710 715 720 Arg Gly His Asp Glu Lys Leu AspGlu Asn Gln His Phe Gly Leu Glu 725 730 735 Ser Gly His Gln His Leu ProTrp Thr Arg Lys Val Tyr Glu Phe Tyr 740 745 750 Ser Ala Pro Ile Val LysPhe Trp Phe Tyr Thr Met Ala Tyr Leu Ala 755 760 765 Phe Leu Met Leu PheThr Tyr Thr Val Leu Val Glu Met Gln Pro Gln 770 775 780 Pro Ser Val GlnGlu Trp Leu Val Ser Ile Tyr Ile Phe Thr Asn Ala 785 790 795 800 Ile GluVal Val Arg Glu Ile Cys Ile Ser Glu Pro Gly Lys Phe Thr 805 810 815 GlnLys Val Lys Val Trp Ile Ser Glu Tyr Trp Asn Leu Thr Glu Thr 820 825 830Val Ala Ile Gly Leu Phe Ser Ala Gly Phe Val Leu Arg Trp Gly Asp 835 840845 Pro Pro Phe His Thr Ala Gly Arg Leu Ile Tyr Cys Ile Asp Ile Ile 850855 860 Phe Trp Phe Ser Arg Leu Leu Asp Phe Phe Ala Val Asn Gln His Ala865 870 875 880 Gly Pro Tyr Val Thr Met Ile Ala Lys Met Thr Ala Asn MetPhe Tyr 885 890 895 Ile Val Ile Ile Met Ala Ile Val Leu Leu Ser Phe GlyVal Ala Arg 900 905 910 Lys Ala Ile Leu Ser Pro Lys Glu Pro Pro Ser TrpSer Leu Ala Arg 915 920 925 Asp Ile Val Phe Glu Pro Tyr Trp Met Ile TyrGly Glu Val Tyr Ala 930 935 940 Gly Glu Ile Asp Val Cys Ser Ser Gln ProSer Cys Pro Pro Gly Ser 945 950 955 960 Phe Leu Thr Pro Phe Leu Gln AlaVal Tyr Leu Phe Val Gln Tyr Ile 965 970 975 Ile Met Val Asn Leu Leu IleAla Phe Phe Asn Asn Val Tyr Leu Asp 980 985 990 Met Glu Ser Ile Ser AsnAsn Leu Trp Lys Tyr Asn Arg Tyr Arg Tyr 995 1000 1005 Ile Met Thr TyrHis Glu Lys Pro Trp Leu Pro Pro Pro Leu Ile 1010 1015 1020 Leu Leu SerHis Val Gly Leu Leu Leu Arg Arg Leu Cys Cys His 1025 1030 1035 Arg AlaPro His Asp Gln Glu Glu Gly Asp Val Gly Leu Lys Leu 1040 1045 1050 TyrLeu Ser Lys Glu Asp Leu Lys Lys Leu His Asp Phe Glu Glu 1055 1060 1065Gln Cys Val Glu Lys Tyr Phe His Glu Lys Met Glu Asp Val Asn 1070 10751080 Cys Ser Cys Glu Glu Arg Ile Arg Val Thr Ser Glu Arg Val Thr 10851090 1095 Glu Met Tyr Phe Gln Leu Lys Glu Met Asn Glu Lys Val Ser Phe1100 1105 1110 Ile Lys Asp Ser Leu Leu Ser Leu Asp Ser Gln Val Gly HisLeu 1115 1120 1125 Gln Asp Leu Ser Ala Leu Thr Val Asp Thr Leu Lys ValLeu Ser 1130 1135 1140 Ala Val Asp Thr Leu Gln Glu Asp Glu Ala Leu LeuAla Lys Arg 1145 1150 1155 Lys His Ser Thr Cys Lys Lys Leu Pro His SerTrp Ser Asn Val 1160 1165 1170 Ile Cys Ala Glu Val Leu Gly Ser Met GluIle Ala Gly Glu Lys 1175 1180 1185 Lys Tyr Gln Tyr Tyr Ser Met Pro SerSer Leu Leu Arg Ser Leu 1190 1195 1200 Ala Gly Gly Arg His Pro Pro ArgVal Gln Arg Gly Ala Leu Leu 1205 1210 1215 Glu Ile Thr Asn Ser Lys ArgGlu Ala Thr Asn Val Arg Asn Asp 1220 1225 1230 Gln Glu Arg Gln Glu ThrGln Ser Ser Ile Val Val Ser Gly Val 1235 1240 1245 Ser Pro Asn Arg GlnAla His Ser Lys Tyr Gly Gln Phe Leu Leu 1250 1255 1260 Val Pro Ser AsnLeu Lys Arg Val Pro Phe Ser Ala Glu Thr Val 1265 1270 1275 Leu Pro LeuSer Arg Pro Ser Val Pro Asp Val Leu Ala Thr Glu 1280 1285 1290 Gln AspIle Gln Thr Glu Val Leu Val His Leu Thr Gly Gln Thr 1295 1300 1305 ProVal Val Ser Asp Trp Ala Ser Val Asp Glu Pro Lys Glu Lys 1310 1315 1320His Glu Pro Ile Ala His Leu Leu Asp Gly Gln Asp Lys Ala Glu 1325 13301335 Gln Val Leu Pro Thr Leu Ser Cys Thr Pro Glu Pro Met Thr Met 13401345 1350 Ser Ser Pro Leu Ser Gln Ala Lys Ile Met Gln Thr Gly Gly Gly1355 1360 1365 Tyr Val Asn Trp Ala Phe Ser Glu Gly Asp Glu Thr Gly ValPhe 1370 1375 1380 Ser Ile Lys Lys Lys Trp Gln Thr Cys Leu Pro Ser ThrCys Asp 1385 1390 1395 Ser Asp Ser Ser Arg Ser Glu Gln His Gln Lys GlnAla Gln Asp 1400 1405 1410 Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser AlaGln Ser Ser Glu 1415 1420 1425 Cys Ser Glu Val Gly Pro Trp Leu Gln ProAsn Thr Ser Phe Trp 1430 1435 1440 Ile Asn Pro Leu Arg Arg Tyr Arg ProPhe Ala Arg Ser His Ser 1445 1450 1455 Phe Arg Phe His Lys Glu Glu LysLeu Met Lys Ile Cys Lys Ile 1460 1465 1470 Lys Asn Leu Ser Gly Ser SerGlu Ile Gly Gln Gly Ala Trp Val 1475 1480 1485 Lys Ala Lys Met Leu ThrLys Asp Arg Arg Leu Ser Lys Lys Lys 1490 1495 1500 Lys Asn Thr Gln GlyLeu Gln Val Pro Ile Ile Thr Val Asn Ala 1505 1510 1515 Cys Ser Gln SerAsp Gln Leu Asn Pro Glu Pro Gly Glu Asn Ser 1520 1525 1530 Ile Ser GluGlu Glu Tyr Ser Lys Asn Trp Phe Thr Val Ser Lys 1535 1540 1545 Phe SerHis Thr Gly Val Glu Pro Tyr Ile His Gln Lys Met Lys 1550 1555 1560 ThrLys Glu Ile Gly Gln Cys Ala Ile Gln Ile Ser Asp Tyr Leu 1565 1570 1575Lys Gln Ser Gln Glu Asp Leu Ser Lys Asn Ser Leu Trp Asn Ser 1580 15851590 Arg Ser Thr Asn Leu Asn Arg Asn Ser Leu Leu Lys Ser Ser Ile 15951600 1605 Gly Val Asp Lys Ile Ser Ala Ser Leu Lys Ser Pro Gln Glu Pro1610 1615 1620 His His His Tyr Ser Ala Ile Glu Arg Asn Asn Leu Met ArgLeu 1625 1630 1635 Ser Gln Thr Ile Pro Phe Thr Pro Val Gln Leu Phe AlaGly Glu 1640 1645 1650 Glu Ile Thr Val Tyr Arg Leu Glu Glu Ser Ser ProLeu Asn Leu 1655 1660 1665 Asp Lys Ser Met Ser Ser Trp Ser Gln Arg GlyArg Ala Ala Met 1670 1675 1680 Ile Gln Val Leu Ser Arg Glu Glu Met AspGly Gly Leu Arg Lys 1685 1690 1695 Ala Met Arg Val Val Ser Thr Trp SerGlu Asp Asp Ile Leu Lys 1700 1705 1710 Pro Gly Gln Val Phe Ile Val LysSer Phe Leu Pro Glu Val Val 1715 1720 1725 Arg Thr Trp His Lys Ile PheGln Glu Ser Thr Val Leu His Leu 1730 1735 1740 Cys Leu Arg Glu Ile GlnGln Gln Arg Ala Ala Gln Lys Leu Ile 1745 1750 1755 Tyr Thr Phe Asn GlnVal Lys Pro Gln Thr Ile Pro Tyr Thr Pro 1760 1765 1770 Arg Phe Leu GluVal Phe Leu Ile Tyr Cys His Ser Ala Asn Gln 1775 1780 1785 Trp Leu ThrIle Glu Lys Tyr Met Thr Gly Glu Phe Arg Lys Tyr 1790 1795 1800 Asn AsnAsn Asn Gly Asp Glu Ile Thr Pro Thr Asn Thr Leu Glu 1805 1810 1815 GluLeu Met Leu Ala Phe Ser His Trp Thr Tyr Glu Tyr Thr Arg 1820 1825 1830Gly Glu Leu Leu Val Leu Asp Leu Gln Gly Val Gly Glu Asn Leu 1835 18401845 Thr Asp Pro Ser Val Ile Lys Pro Glu Val Lys Gln Ser Arg Gly 18501855 1860 Met Val Phe Gly Pro Ala Asn Leu Gly Glu Asp Ala Ile Arg Asn1865 1870 1875 Phe Ile Ala Lys His His Cys Asn Ser Cys Cys Arg Lys LeuLys 1880 1885 1890 Leu Pro Asp Leu Lys Arg Asn Asp Tyr Ser Pro Glu ArgIle Asn 1895 1900 1905 Ser Thr Phe Gly Leu Glu Ile Lys Ile Glu Ser AlaGlu Glu Pro 1910 1915 1920 Pro Ala Arg Glu Thr Gly Arg Asn Ser Pro GluAsp Asp Met Gln 1925 1930 1935 Leu 7 5925 DNA Homo sapiens CDS(1)..(5922) 7 atg att atc cta tct aag tcc cag aaa tcc tgg att aaa ggagta ttt 48 Met Ile Ile Leu Ser Lys Ser Gln Lys Ser Trp Ile Lys Gly ValPhe 1 5 10 15 gac aag aga gaa tgt agc aca atc ata ccc agc tca aaa aatcct cac 96 Asp Lys Arg Glu Cys Ser Thr Ile Ile Pro Ser Ser Lys Asn ProHis 20 25 30 aga tgt act cca gta tgc caa gtc tgc cag aat tta atc agg tgttac 144 Arg Cys Thr Pro Val Cys Gln Val Cys Gln Asn Leu Ile Arg Cys Tyr35 40 45 tgt ggc cga ctg att gga gac cat gct ggg ata gat tat tcc tgg acc192 Cys Gly Arg Leu Ile Gly Asp His Ala Gly Ile Asp Tyr Ser Trp Thr 5055 60 atc tca gct gcc aag ggt aaa gaa agt gaa caa tgg tct gtt gaa aag240 Ile Ser Ala Ala Lys Gly Lys Glu Ser Glu Gln Trp Ser Val Glu Lys 6570 75 80 cac aca acg aaa agc cca aca gat act ttt ggc acg att aat ttc caa288 His Thr Thr Lys Ser Pro Thr Asp Thr Phe Gly Thr Ile Asn Phe Gln 8590 95 gat gga gag cac acc cat cat gcc aag tat att aga act tct tat gat336 Asp Gly Glu His Thr His His Ala Lys Tyr Ile Arg Thr Ser Tyr Asp 100105 110 aca aaa ctg gat cat ctg tta cat tta atg ttg aaa gag tgg aaa atg384 Thr Lys Leu Asp His Leu Leu His Leu Met Leu Lys Glu Trp Lys Met 115120 125 gaa ctg ccc aag ctt gtg atc tca gtc cat ggg ggc atc cag aac ttt432 Glu Leu Pro Lys Leu Val Ile Ser Val His Gly Gly Ile Gln Asn Phe 130135 140 act atg ccc tct aaa ttt aaa gag att ttc agc caa ggt ttg gtt aaa480 Thr Met Pro Ser Lys Phe Lys Glu Ile Phe Ser Gln Gly Leu Val Lys 145150 155 160 gct gca gag aca aca gga gcg tgg ata ata act gaa ggc atc aataca 528 Ala Ala Glu Thr Thr Gly Ala Trp Ile Ile Thr Glu Gly Ile Asn Thr165 170 175 gga gtg tcc aag cat gtt ggg gat gcc ttg aaa tcc cat tcc tctcat 576 Gly Val Ser Lys His Val Gly Asp Ala Leu Lys Ser His Ser Ser His180 185 190 tcc ttg aga aaa atc tgg aca gtt gga atc cct cct tgg ggt gtcatt 624 Ser Leu Arg Lys Ile Trp Thr Val Gly Ile Pro Pro Trp Gly Val Ile195 200 205 gag aac cag aga gac ctt att gga aaa gat gtg gtg tgc ctg taccag 672 Glu Asn Gln Arg Asp Leu Ile Gly Lys Asp Val Val Cys Leu Tyr Gln210 215 220 act ctg gat aac ccc ctc agc aag ctc aca aca ctc aac agc atgcac 720 Thr Leu Asp Asn Pro Leu Ser Lys Leu Thr Thr Leu Asn Ser Met His225 230 235 240 tcg cac ttc atc ctg tct gat gat ggg acc gtg ggc aag tatgga aat 768 Ser His Phe Ile Leu Ser Asp Asp Gly Thr Val Gly Lys Tyr GlyAsn 245 250 255 gaa atg aag ctc aga agg aac ctg gag aag tac ctc tct ctgcag aaa 816 Glu Met Lys Leu Arg Arg Asn Leu Glu Lys Tyr Leu Ser Leu GlnLys 260 265 270 ata cac tgc cgc tca aga caa ggc gtg ccg gtc gtg ggg ctggtg gtg 864 Ile His Cys Arg Ser Arg Gln Gly Val Pro Val Val Gly Leu ValVal 275 280 285 gaa ggc ggt ccc aac gtc atc ctg tca gtg tgg gag act gtcaag gac 912 Glu Gly Gly Pro Asn Val Ile Leu Ser Val Trp Glu Thr Val LysAsp 290 295 300 aag gac cca gtg gtg gtg tgt gag ggc aca ggt agg gcg gctgac ctc 960 Lys Asp Pro Val Val Val Cys Glu Gly Thr Gly Arg Ala Ala AspLeu 305 310 315 320 ctg gcc ttc aca cac aaa cac ctg gca gat gaa ggg atgctg cga cct 1008 Leu Ala Phe Thr His Lys His Leu Ala Asp Glu Gly Met LeuArg Pro 325 330 335 cag gtg aaa gag gag atc atc tgc atg att cag aac actttc aac ttt 1056 Gln Val Lys Glu Glu Ile Ile Cys Met Ile Gln Asn Thr PheAsn Phe 340 345 350 agt ctt aaa cag tcc aag cac ctt ttc caa att cta atggag tgt atg 1104 Ser Leu Lys Gln Ser Lys His Leu Phe Gln Ile Leu Met GluCys Met 355 360 365 gtt cac agg gat tgt att acc ata ttt gat gct gac tctgaa gag cag 1152 Val His Arg Asp Cys Ile Thr Ile Phe Asp Ala Asp Ser GluGlu Gln 370 375 380 caa gac ctg gac tta gca atc cta aca gct ttg ctg aagggc aca aat 1200 Gln Asp Leu Asp Leu Ala Ile Leu Thr Ala Leu Leu Lys GlyThr Asn 385 390 395 400 tta tca gcg tca gag caa tta aat ctg gca atg gcttgg gac agg gtg 1248 Leu Ser Ala Ser Glu Gln Leu Asn Leu Ala Met Ala TrpAsp Arg Val 405 410 415 gac att gcc aag aaa cat atc cta att tat gaa caacac tgg aag cct 1296 Asp Ile Ala Lys Lys His Ile Leu Ile Tyr Glu Gln HisTrp Lys Pro 420 425 430 gat gcc ctg gaa caa gca atg tca gat gct tta gtgatg gat cgg gtg 1344 Asp Ala Leu Glu Gln Ala Met Ser Asp Ala Leu Val MetAsp Arg Val 435 440 445 gat ttt gtg aag ctc tta ata gaa tat gga gtg aacctc cat cgc ttt 1392 Asp Phe Val Lys Leu Leu Ile Glu Tyr Gly Val Asn LeuHis Arg Phe 450 455 460 ctt acc atc cct cga ctg gaa gag ctc tac aat acaaaa caa gga cct 1440 Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr LysGln Gly Pro 465 470 475 480 act aat aca ctc ttg cat cat ctc gtc caa gatgtg aaa cag cat acc 1488 Thr Asn Thr Leu Leu His His Leu Val Gln Asp ValLys Gln His Thr 485 490 495 ctt ctt tca ggc tac cga ata acc ttg att gacatt gga tta gta gta 1536 Leu Leu Ser Gly Tyr Arg Ile Thr Leu Ile Asp IleGly Leu Val Val 500 505 510 gaa tac ctc att ggt aga gca tat cgc agc aactac act aga aaa cat 1584 Glu Tyr Leu Ile Gly Arg Ala Tyr Arg Ser Asn TyrThr Arg Lys His 515 520 525 ttc aga gcc ctc tac aac aac ctc tac aga aaatac aag cac cag aga 1632 Phe Arg Ala Leu Tyr Asn Asn Leu Tyr Arg Lys TyrLys His Gln Arg 530 535 540 cac tcc tca gga aat aga aat gag tct gca gaaagt acg ctg cac tcc 1680 His Ser Ser Gly Asn Arg Asn Glu Ser Ala Glu SerThr Leu His Ser 545 550 555 560 cag ttc att aga act gca cag cca tac aaattc aag gaa aag tct ata 1728 Gln Phe Ile Arg Thr Ala Gln Pro Tyr Lys PheLys Glu Lys Ser Ile 565 570 575 gtc ctt cat aaa tca agg aag aag tca aaagaa caa aat gta tca gat 1776 Val Leu His Lys Ser Arg Lys Lys Ser Lys GluGln Asn Val Ser Asp 580 585 590 gac cct gag tct act ggc ttt ctt tac ccttac aat gac ctg ctg gtt 1824 Asp Pro Glu Ser Thr Gly Phe Leu Tyr Pro TyrAsn Asp Leu Leu Val 595 600 605 tgg gct gtg ctg atg aaa agg cag aag atggct atg ttc ttc tgg cag 1872 Trp Ala Val Leu Met Lys Arg Gln Lys Met AlaMet Phe Phe Trp Gln 610 615 620 cat gga gag gag gcc acg gtt aaa gcc gtgatt gcg tgt atc ctc tac 1920 His Gly Glu Glu Ala Thr Val Lys Ala Val IleAla Cys Ile Leu Tyr 625 630 635 640 cgg gca atg gcc cat gaa gct aag gagagt cac atg gtg gat gat gcc 1968 Arg Ala Met Ala His Glu Ala Lys Glu SerHis Met Val Asp Asp Ala 645 650 655 tca gaa gag ttg aag aat tac tca aaacag ttt ggc cag ctg gct ctg 2016 Ser Glu Glu Leu Lys Asn Tyr Ser Lys GlnPhe Gly Gln Leu Ala Leu 660 665 670 gac ttg ttg gag aag gca ttc aag cagaat gag cgc atg gcc atg acg 2064 Asp Leu Leu Glu Lys Ala Phe Lys Gln AsnGlu Arg Met Ala Met Thr 675 680 685 ctg ttg acg tat gaa ctc agg aac tggagc aat tcg acc tgc ctg aaa 2112 Leu Leu Thr Tyr Glu Leu Arg Asn Trp SerAsn Ser Thr Cys Leu Lys 690 695 700 ctg gcc gtg tcg gga gga tta cga cccttt gtt tca cat act tgt acc 2160 Leu Ala Val Ser Gly Gly Leu Arg Pro PheVal Ser His Thr Cys Thr 705 710 715 720 cag atg cta ctg aca gac atg tggatg ggg agg ctg aaa atg agg aaa 2208 Gln Met Leu Leu Thr Asp Met Trp MetGly Arg Leu Lys Met Arg Lys 725 730 735 aac tct tgg tta aag att att ataagc att att tta cca ccc acc att 2256 Asn Ser Trp Leu Lys Ile Ile Ile SerIle Ile Leu Pro Pro Thr Ile 740 745 750 ttg aca ctg gaa ttt aaa agc aaagct gag atg tca cat gtt ccc cag 2304 Leu Thr Leu Glu Phe Lys Ser Lys AlaGlu Met Ser His Val Pro Gln 755 760 765 tcc cag gac ttc caa ttt atg tggtat tac agt gac cag aac gcc agc 2352 Ser Gln Asp Phe Gln Phe Met Trp TyrTyr Ser Asp Gln Asn Ala Ser 770 775 780 agt tcc aaa gaa agt gct tct gtgaaa gag tat gat ttg gaa agg ggc 2400 Ser Ser Lys Glu Ser Ala Ser Val LysGlu Tyr Asp Leu Glu Arg Gly 785 790 795 800 cat gat gag aaa ctg gat gaaaat cag cat ttt ggt ttg gaa agt ggg 2448 His Asp Glu Lys Leu Asp Glu AsnGln His Phe Gly Leu Glu Ser Gly 805 810 815 cac caa cac ctt ccg tgg accagg aaa gtc tat gag ttc tac agt gct 2496 His Gln His Leu Pro Trp Thr ArgLys Val Tyr Glu Phe Tyr Ser Ala 820 825 830 cca att gtc aag ttt tgg ttttat acg atc tgt att tca gaa cct ggg 2544 Pro Ile Val Lys Phe Trp Phe TyrThr Ile Cys Ile Ser Glu Pro Gly 835 840 845 aag ttt acc caa aag gtg aaggta tgg att agt gag tac tgg aac tta 2592 Lys Phe Thr Gln Lys Val Lys ValTrp Ile Ser Glu Tyr Trp Asn Leu 850 855 860 aca gaa act gtg gcc att ggcctg ttt tca gct ggc ttc gtc ctt cga 2640 Thr Glu Thr Val Ala Ile Gly LeuPhe Ser Ala Gly Phe Val Leu Arg 865 870 875 880 tgg ggt gac cct cct tttcac aca gcg gga aga ctg atc tac tgc ata 2688 Trp Gly Asp Pro Pro Phe HisThr Ala Gly Arg Leu Ile Tyr Cys Ile 885 890 895 gac atc ata ttc tgg ttctca cgg ctc ctg gac ttc ttt gct gtg aat 2736 Asp Ile Ile Phe Trp Phe SerArg Leu Leu Asp Phe Phe Ala Val Asn 900 905 910 caa cat gca ggt cca tatgtg acc atg att gca aaa atg aca gca aac 2784 Gln His Ala Gly Pro Tyr ValThr Met Ile Ala Lys Met Thr Ala Asn 915 920 925 atg ttc tat att gtg atcatc atg gcc ata gtc ctg ctg agc ttt gga 2832 Met Phe Tyr Ile Val Ile IleMet Ala Ile Val Leu Leu Ser Phe Gly 930 935 940 gtg gca cgc aag gcc atcctt tcg cca aaa gag cca cca tct tgg agt 2880 Val Ala Arg Lys Ala Ile LeuSer Pro Lys Glu Pro Pro Ser Trp Ser 945 950 955 960 cta gct cga gat attgta ttt gag cca tac tgg atg ata tac gga gaa 2928 Leu Ala Arg Asp Ile ValPhe Glu Pro Tyr Trp Met Ile Tyr Gly Glu 965 970 975 gtc tat gct gga gaaata gat gtt tgt tca agc cag cca tcc tgc cct 2976 Val Tyr Ala Gly Glu IleAsp Val Cys Ser Ser Gln Pro Ser Cys Pro 980 985 990 cct ggt tct ttt cttact cca ttc ttg caa gct gtc tac ctc ttc gtg 3024 Pro Gly Ser Phe Leu ThrPro Phe Leu Gln Ala Val Tyr Leu Phe Val 995 1000 1005 caa tat atc atcatg gtg aac ctg ttg att gct ttc ttc aac aac 3069 Gln Tyr Ile Ile Met ValAsn Leu Leu Ile Ala Phe Phe Asn Asn 1010 1015 1020 gtt tac tta gat atggaa tcc att tca aat aac ctg tgg aaa tac 3114 Val Tyr Leu Asp Met Glu SerIle Ser Asn Asn Leu Trp Lys Tyr 1025 1030 1035 aac cgc tat cgc tac atcatg acc tac cac gag aag ccc tgg ctg 3159 Asn Arg Tyr Arg Tyr Ile Met ThrTyr His Glu Lys Pro Trp Leu 1040 1045 1050 ccc cca cct ctc atc ctg ctgagc cac gtg ggc ctt ctc ctc cgc 3204 Pro Pro Pro Leu Ile Leu Leu Ser HisVal Gly Leu Leu Leu Arg 1055 1060 1065 cgc ctg tgc tgt cat cga gct cctcac gac caa gaa gag ggt gac 3249 Arg Leu Cys Cys His Arg Ala Pro His AspGln Glu Glu Gly Asp 1070 1075 1080 gtt gga tta aaa ctc tac ctc agt aaggag gat ctg aaa aaa ctt 3294 Val Gly Leu Lys Leu Tyr Leu Ser Lys Glu AspLeu Lys Lys Leu 1085 1090 1095 cat gat ttt gag gag cag tgc gtg gaa aaatac ttc cat gag aag 3339 His Asp Phe Glu Glu Gln Cys Val Glu Lys Tyr PheHis Glu Lys 1100 1105 1110 atg gaa gat gtg aat tgt agt tgt gag gaa cgaatc cga gtg aca 3384 Met Glu Asp Val Asn Cys Ser Cys Glu Glu Arg Ile ArgVal Thr 1115 1120 1125 tca gaa agg gtt aca gag atg tac ttc cag ctg aaagaa atg aat 3429 Ser Glu Arg Val Thr Glu Met Tyr Phe Gln Leu Lys Glu MetAsn 1130 1135 1140 gaa aag gtg tct ttt ata aag gac tcc tta ctg tct ttggac agc 3474 Glu Lys Val Ser Phe Ile Lys Asp Ser Leu Leu Ser Leu Asp Ser1145 1150 1155 cag gtg gga cac ctg cag gat ctc tct gcc ctg act gtg gatacc 3519 Gln Val Gly His Leu Gln Asp Leu Ser Ala Leu Thr Val Asp Thr1160 1165 1170 ctg aaa gtc ctt tct gct gtt gac act ttg caa gag gat gaggct 3564 Leu Lys Val Leu Ser Ala Val Asp Thr Leu Gln Glu Asp Glu Ala1175 1180 1185 ctc ctg gcc aag aga aag cat tct act tgc aaa aaa ctt ccccac 3609 Leu Leu Ala Lys Arg Lys His Ser Thr Cys Lys Lys Leu Pro His1190 1195 1200 agc tgg agc aat gtc atc tgt gca gag gtt cta ggc agc atggag 3654 Ser Trp Ser Asn Val Ile Cys Ala Glu Val Leu Gly Ser Met Glu1205 1210 1215 atc gct gga gag aag aaa tac cag tat tat agc atg ccc tcttct 3699 Ile Ala Gly Glu Lys Lys Tyr Gln Tyr Tyr Ser Met Pro Ser Ser1220 1225 1230 ttg ctg agg agc ctg gct gga ggc cgg cat ccc cca aga gtgcag 3744 Leu Leu Arg Ser Leu Ala Gly Gly Arg His Pro Pro Arg Val Gln1235 1240 1245 agg ggg gca ctt ctt gag att aca aac agt aaa aga gag gctaca 3789 Arg Gly Ala Leu Leu Glu Ile Thr Asn Ser Lys Arg Glu Ala Thr1250 1255 1260 aat gta aga aat gac cag gaa agg caa gaa aca caa agt agtata 3834 Asn Val Arg Asn Asp Gln Glu Arg Gln Glu Thr Gln Ser Ser Ile1265 1270 1275 gtg gtt tct ggg gtg tct cct aac agg caa gca cac tca aagtat 3879 Val Val Ser Gly Val Ser Pro Asn Arg Gln Ala His Ser Lys Tyr1280 1285 1290 ggc cag ttt ctt ctg gtc ccc tct aat cta aag cga gtt cctttt 3924 Gly Gln Phe Leu Leu Val Pro Ser Asn Leu Lys Arg Val Pro Phe1295 1300 1305 tca gca gaa act gtc ttg cct ctg tcc aga ccc tct gtg ccagat 3969 Ser Ala Glu Thr Val Leu Pro Leu Ser Arg Pro Ser Val Pro Asp1310 1315 1320 gtg ctg gca act gaa cag gac atc cag act gag gtt ctt gttcat 4014 Val Leu Ala Thr Glu Gln Asp Ile Gln Thr Glu Val Leu Val His1325 1330 1335 ctg act ggg cag acc cca gtt gtc tct gac tgg gca tca gtggat 4059 Leu Thr Gly Gln Thr Pro Val Val Ser Asp Trp Ala Ser Val Asp1340 1345 1350 gaa ccc aag gaa aag cac gag cct att gct cac tta ctg gatgga 4104 Glu Pro Lys Glu Lys His Glu Pro Ile Ala His Leu Leu Asp Gly1355 1360 1365 caa gac aag gca gag caa gtg cta ccc act ttg agt tgc acacct 4149 Gln Asp Lys Ala Glu Gln Val Leu Pro Thr Leu Ser Cys Thr Pro1370 1375 1380 gaa ccc atg aca atg agc tcc cct ctt tcc caa gcc aag atcatg 4194 Glu Pro Met Thr Met Ser Ser Pro Leu Ser Gln Ala Lys Ile Met1385 1390 1395 caa act gga ggt gga tat gta aac tgg gca ttt tca gaa ggtgat 4239 Gln Thr Gly Gly Gly Tyr Val Asn Trp Ala Phe Ser Glu Gly Asp1400 1405 1410 gaa act ggt gtg ttt agc atc aag aaa aag tgg caa acc tgcttg 4284 Glu Thr Gly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr Cys Leu1415 1420 1425 ccc tcc act tgt gac agt gat tcc tct cgg agt gaa cag caccag 4329 Pro Ser Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln1430 1435 1440 aag cag gcc cag gac agc tcc cta tct gat aac tca aca agatcg 4374 Lys Gln Ala Gln Asp Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser1445 1450 1455 gcc cag agt agt gaa tgc tca gag gtg gga cca tgg ctt cagcca 4419 Ala Gln Ser Ser Glu Cys Ser Glu Val Gly Pro Trp Leu Gln Pro1460 1465 1470 aac aca tcc ttt tgg atc aat cct ctc cgc aga tac agg cccttc 4464 Asn Thr Ser Phe Trp Ile Asn Pro Leu Arg Arg Tyr Arg Pro Phe1475 1480 1485 gct agg agt cat agt ttt aga ttc cat aag gag gag aaa ttgatg 4509 Ala Arg Ser His Ser Phe Arg Phe His Lys Glu Glu Lys Leu Met1490 1495 1500 aag atc tgt aag att aaa aat ctt tca ggc tct tca gaa ataggg 4554 Lys Ile Cys Lys Ile Lys Asn Leu Ser Gly Ser Ser Glu Ile Gly1505 1510 1515 cag gga gca tgg gtc aaa gcg aaa atg cta acc aaa gac aggaga 4599 Gln Gly Ala Trp Val Lys Ala Lys Met Leu Thr Lys Asp Arg Arg1520 1525 1530 ctg tca aag aaa aag aag aat act caa gga ctc cag gtg ccaatc 4644 Leu Ser Lys Lys Lys Lys Asn Thr Gln Gly Leu Gln Val Pro Ile1535 1540 1545 ata aca gtc aat gcc tgc tct cag agt gac cag ttg aat ccagag 4689 Ile Thr Val Asn Ala Cys Ser Gln Ser Asp Gln Leu Asn Pro Glu1550 1555 1560 cca gga gaa aac agc atc tct gaa gag gag tac agc aag aactgg 4734 Pro Gly Glu Asn Ser Ile Ser Glu Glu Glu Tyr Ser Lys Asn Trp1565 1570 1575 ttc aca gtg tcc aaa ttt agt cac aca ggt gta gaa cct tacata 4779 Phe Thr Val Ser Lys Phe Ser His Thr Gly Val Glu Pro Tyr Ile1580 1585 1590 cat cag aaa atg aaa act aaa gaa att gga caa tgt gct atacaa 4824 His Gln Lys Met Lys Thr Lys Glu Ile Gly Gln Cys Ala Ile Gln1595 1600 1605 atc agt gat tac cta aag cag tct caa gag gat ctc agc aaaaac 4869 Ile Ser Asp Tyr Leu Lys Gln Ser Gln Glu Asp Leu Ser Lys Asn1610 1615 1620 tct ttg tgg aat tcc agg agc acc aac ctc aat agg aac tccctg 4914 Ser Leu Trp Asn Ser Arg Ser Thr Asn Leu Asn Arg Asn Ser Leu1625 1630 1635 ctg aaa agt tca att gga gtt gac aag atc tca gcc tcc ttaaaa 4959 Leu Lys Ser Ser Ile Gly Val Asp Lys Ile Ser Ala Ser Leu Lys1640 1645 1650 agc cct caa gag cct cac cat cat tat tca gcc att gaa aggaat 5004 Ser Pro Gln Glu Pro His His His Tyr Ser Ala Ile Glu Arg Asn1655 1660 1665 aat tta atg agg ctt tct cag acc ata cca ttt aca cca gtccaa 5049 Asn Leu Met Arg Leu Ser Gln Thr Ile Pro Phe Thr Pro Val Gln1670 1675 1680 ctg ttt gca gga gaa gaa ata act gtc tac agg ttg gag gagagt 5094 Leu Phe Ala Gly Glu Glu Ile Thr Val Tyr Arg Leu Glu Glu Ser1685 1690 1695 tcc cct tta aac ctt gat aaa agc atg tcc tct tgg tct cagcgt 5139 Ser Pro Leu Asn Leu Asp Lys Ser Met Ser Ser Trp Ser Gln Arg1700 1705 1710 ggg aga gcg gca atg atc cag gta ttg tcc cga gag gag atggat 5184 Gly Arg Ala Ala Met Ile Gln Val Leu Ser Arg Glu Glu Met Asp1715 1720 1725 ggg ggc ctc cgt aaa gct atg aga gtc gtc agc act tgg tctgag 5229 Gly Gly Leu Arg Lys Ala Met Arg Val Val Ser Thr Trp Ser Glu1730 1735 1740 gat gac att ctc aag ccg gga caa gtt ttc att gtc aag tccttt 5274 Asp Asp Ile Leu Lys Pro Gly Gln Val Phe Ile Val Lys Ser Phe1745 1750 1755 ctt cct gag gtt gtg cgg aca tgg cat aaa atc ttc cag gagagc 5319 Leu Pro Glu Val Val Arg Thr Trp His Lys Ile Phe Gln Glu Ser1760 1765 1770 act gtg ctt cat ctt tgc ctc agg gaa att caa caa caa agagct 5364 Thr Val Leu His Leu Cys Leu Arg Glu Ile Gln Gln Gln Arg Ala1775 1780 1785 gct caa aaa ttg atc tat acc ttc aac caa gtg aaa cca caaacc 5409 Ala Gln Lys Leu Ile Tyr Thr Phe Asn Gln Val Lys Pro Gln Thr1790 1795 1800 ata ccc tac aca cca agg ttc ctg gaa gtt ttc tta atc tactgc 5454 Ile Pro Tyr Thr Pro Arg Phe Leu Glu Val Phe Leu Ile Tyr Cys1805 1810 1815 cat tca gcc aac cag tgg ttg acc att gag aag tat atg acaggg 5499 His Ser Ala Asn Gln Trp Leu Thr Ile Glu Lys Tyr Met Thr Gly1820 1825 1830 gag ttc cgg aag tat aac aac aac aat ggt gat gaa atc accccc 5544 Glu Phe Arg Lys Tyr Asn Asn Asn Asn Gly Asp Glu Ile Thr Pro1835 1840 1845 acc aac acc ctg gag gag ctg atg ttg gct ttc tct cac tggacc 5589 Thr Asn Thr Leu Glu Glu Leu Met Leu Ala Phe Ser His Trp Thr1850 1855 1860 tat gag tac act cgg gga gag ctg ctg gtt tta gat ttg caaggt 5634 Tyr Glu Tyr Thr Arg Gly Glu Leu Leu Val Leu Asp Leu Gln Gly1865 1870 1875 gtt gga gaa aat ttg aca gat cca tct gtt ata aaa cct gaagtc 5679 Val Gly Glu Asn Leu Thr Asp Pro Ser Val Ile Lys Pro Glu Val1880 1885 1890 aaa caa tca aga gga atg gtg ttt gga ccg gcc aat ttg ggggaa 5724 Lys Gln Ser Arg Gly Met Val Phe Gly Pro Ala Asn Leu Gly Glu1895 1900 1905 gat gca att aga aac ttc att gca aaa cat cat tgt aac tcctgc 5769 Asp Ala Ile Arg Asn Phe Ile Ala Lys His His Cys Asn Ser Cys1910 1915 1920 tgc cgg aag ctc aaa ctc ccg gat tta aaa aga aat gac tattcc 5814 Cys Arg Lys Leu Lys Leu Pro Asp Leu Lys Arg Asn Asp Tyr Ser1925 1930 1935 cct gaa agg ata aat tcc acc ttt gga ctt gag ata aaa atagaa 5859 Pro Glu Arg Ile Asn Ser Thr Phe Gly Leu Glu Ile Lys Ile Glu1940 1945 1950 tca gct gag gag cct cca gca agg gag acg ggt aga aat tcccca 5904 Ser Ala Glu Glu Pro Pro Ala Arg Glu Thr Gly Arg Asn Ser Pro1955 1960 1965 gaa gat gat atg caa cta taa 5925 Glu Asp Asp Met Gln Leu1970 8 1974 PRT Homo sapiens 8 Met Ile Ile Leu Ser Lys Ser Gln Lys SerTrp Ile Lys Gly Val Phe 1 5 10 15 Asp Lys Arg Glu Cys Ser Thr Ile IlePro Ser Ser Lys Asn Pro His 20 25 30 Arg Cys Thr Pro Val Cys Gln Val CysGln Asn Leu Ile Arg Cys Tyr 35 40 45 Cys Gly Arg Leu Ile Gly Asp His AlaGly Ile Asp Tyr Ser Trp Thr 50 55 60 Ile Ser Ala Ala Lys Gly Lys Glu SerGlu Gln Trp Ser Val Glu Lys 65 70 75 80 His Thr Thr Lys Ser Pro Thr AspThr Phe Gly Thr Ile Asn Phe Gln 85 90 95 Asp Gly Glu His Thr His His AlaLys Tyr Ile Arg Thr Ser Tyr Asp 100 105 110 Thr Lys Leu Asp His Leu LeuHis Leu Met Leu Lys Glu Trp Lys Met 115 120 125 Glu Leu Pro Lys Leu ValIle Ser Val His Gly Gly Ile Gln Asn Phe 130 135 140 Thr Met Pro Ser LysPhe Lys Glu Ile Phe Ser Gln Gly Leu Val Lys 145 150 155 160 Ala Ala GluThr Thr Gly Ala Trp Ile Ile Thr Glu Gly Ile Asn Thr 165 170 175 Gly ValSer Lys His Val Gly Asp Ala Leu Lys Ser His Ser Ser His 180 185 190 SerLeu Arg Lys Ile Trp Thr Val Gly Ile Pro Pro Trp Gly Val Ile 195 200 205Glu Asn Gln Arg Asp Leu Ile Gly Lys Asp Val Val Cys Leu Tyr Gln 210 215220 Thr Leu Asp Asn Pro Leu Ser Lys Leu Thr Thr Leu Asn Ser Met His 225230 235 240 Ser His Phe Ile Leu Ser Asp Asp Gly Thr Val Gly Lys Tyr GlyAsn 245 250 255 Glu Met Lys Leu Arg Arg Asn Leu Glu Lys Tyr Leu Ser LeuGln Lys 260 265 270 Ile His Cys Arg Ser Arg Gln Gly Val Pro Val Val GlyLeu Val Val 275 280 285 Glu Gly Gly Pro Asn Val Ile Leu Ser Val Trp GluThr Val Lys Asp 290 295 300 Lys Asp Pro Val Val Val Cys Glu Gly Thr GlyArg Ala Ala Asp Leu 305 310 315 320 Leu Ala Phe Thr His Lys His Leu AlaAsp Glu Gly Met Leu Arg Pro 325 330 335 Gln Val Lys Glu Glu Ile Ile CysMet Ile Gln Asn Thr Phe Asn Phe 340 345 350 Ser Leu Lys Gln Ser Lys HisLeu Phe Gln Ile Leu Met Glu Cys Met 355 360 365 Val His Arg Asp Cys IleThr Ile Phe Asp Ala Asp Ser Glu Glu Gln 370 375 380 Gln Asp Leu Asp LeuAla Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn 385 390 395 400 Leu Ser AlaSer Glu Gln Leu Asn Leu Ala Met Ala Trp Asp Arg Val 405 410 415 Asp IleAla Lys Lys His Ile Leu Ile Tyr Glu Gln His Trp Lys Pro 420 425 430 AspAla Leu Glu Gln Ala Met Ser Asp Ala Leu Val Met Asp Arg Val 435 440 445Asp Phe Val Lys Leu Leu Ile Glu Tyr Gly Val Asn Leu His Arg Phe 450 455460 Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr Lys Gln Gly Pro 465470 475 480 Thr Asn Thr Leu Leu His His Leu Val Gln Asp Val Lys Gln HisThr 485 490 495 Leu Leu Ser Gly Tyr Arg Ile Thr Leu Ile Asp Ile Gly LeuVal Val 500 505 510 Glu Tyr Leu Ile Gly Arg Ala Tyr Arg Ser Asn Tyr ThrArg Lys His 515 520 525 Phe Arg Ala Leu Tyr Asn Asn Leu Tyr Arg Lys TyrLys His Gln Arg 530 535 540 His Ser Ser Gly Asn Arg Asn Glu Ser Ala GluSer Thr Leu His Ser 545 550 555 560 Gln Phe Ile Arg Thr Ala Gln Pro TyrLys Phe Lys Glu Lys Ser Ile 565 570 575 Val Leu His Lys Ser Arg Lys LysSer Lys Glu Gln Asn Val Ser Asp 580 585 590 Asp Pro Glu Ser Thr Gly PheLeu Tyr Pro Tyr Asn Asp Leu Leu Val 595 600 605 Trp Ala Val Leu Met LysArg Gln Lys Met Ala Met Phe Phe Trp Gln 610 615 620 His Gly Glu Glu AlaThr Val Lys Ala Val Ile Ala Cys Ile Leu Tyr 625 630 635 640 Arg Ala MetAla His Glu Ala Lys Glu Ser His Met Val Asp Asp Ala 645 650 655 Ser GluGlu Leu Lys Asn Tyr Ser Lys Gln Phe Gly Gln Leu Ala Leu 660 665 670 AspLeu Leu Glu Lys Ala Phe Lys Gln Asn Glu Arg Met Ala Met Thr 675 680 685Leu Leu Thr Tyr Glu Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys 690 695700 Leu Ala Val Ser Gly Gly Leu Arg Pro Phe Val Ser His Thr Cys Thr 705710 715 720 Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Lys Met ArgLys 725 730 735 Asn Ser Trp Leu Lys Ile Ile Ile Ser Ile Ile Leu Pro ProThr Ile 740 745 750 Leu Thr Leu Glu Phe Lys Ser Lys Ala Glu Met Ser HisVal Pro Gln 755 760 765 Ser Gln Asp Phe Gln Phe Met Trp Tyr Tyr Ser AspGln Asn Ala Ser 770 775 780 Ser Ser Lys Glu Ser Ala Ser Val Lys Glu TyrAsp Leu Glu Arg Gly 785 790 795 800 His Asp Glu Lys Leu Asp Glu Asn GlnHis Phe Gly Leu Glu Ser Gly 805 810 815 His Gln His Leu Pro Trp Thr ArgLys Val Tyr Glu Phe Tyr Ser Ala 820 825 830 Pro Ile Val Lys Phe Trp PheTyr Thr Ile Cys Ile Ser Glu Pro Gly 835 840 845 Lys Phe Thr Gln Lys ValLys Val Trp Ile Ser Glu Tyr Trp Asn Leu 850 855 860 Thr Glu Thr Val AlaIle Gly Leu Phe Ser Ala Gly Phe Val Leu Arg 865 870 875 880 Trp Gly AspPro Pro Phe His Thr Ala Gly Arg Leu Ile Tyr Cys Ile 885 890 895 Asp IleIle Phe Trp Phe Ser Arg Leu Leu Asp Phe Phe Ala Val Asn 900 905 910 GlnHis Ala Gly Pro Tyr Val Thr Met Ile Ala Lys Met Thr Ala Asn 915 920 925Met Phe Tyr Ile Val Ile Ile Met Ala Ile Val Leu Leu Ser Phe Gly 930 935940 Val Ala Arg Lys Ala Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp Ser 945950 955 960 Leu Ala Arg Asp Ile Val Phe Glu Pro Tyr Trp Met Ile Tyr GlyGlu 965 970 975 Val Tyr Ala Gly Glu Ile Asp Val Cys Ser Ser Gln Pro SerCys Pro 980 985 990 Pro Gly Ser Phe Leu Thr Pro Phe Leu Gln Ala Val TyrLeu Phe Val 995 1000 1005 Gln Tyr Ile Ile Met Val Asn Leu Leu Ile AlaPhe Phe Asn Asn 1010 1015 1020 Val Tyr Leu Asp Met Glu Ser Ile Ser AsnAsn Leu Trp Lys Tyr 1025 1030 1035 Asn Arg Tyr Arg Tyr Ile Met Thr TyrHis Glu Lys Pro Trp Leu 1040 1045 1050 Pro Pro Pro Leu Ile Leu Leu SerHis Val Gly Leu Leu Leu Arg 1055 1060 1065 Arg Leu Cys Cys His Arg AlaPro His Asp Gln Glu Glu Gly Asp 1070 1075 1080 Val Gly Leu Lys Leu TyrLeu Ser Lys Glu Asp Leu Lys Lys Leu 1085 1090 1095 His Asp Phe Glu GluGln Cys Val Glu Lys Tyr Phe His Glu Lys 1100 1105 1110 Met Glu Asp ValAsn Cys Ser Cys Glu Glu Arg Ile Arg Val Thr 1115 1120 1125 Ser Glu ArgVal Thr Glu Met Tyr Phe Gln Leu Lys Glu Met Asn 1130 1135 1140 Glu LysVal Ser Phe Ile Lys Asp Ser Leu Leu Ser Leu Asp Ser 1145 1150 1155 GlnVal Gly His Leu Gln Asp Leu Ser Ala Leu Thr Val Asp Thr 1160 1165 1170Leu Lys Val Leu Ser Ala Val Asp Thr Leu Gln Glu Asp Glu Ala 1175 11801185 Leu Leu Ala Lys Arg Lys His Ser Thr Cys Lys Lys Leu Pro His 11901195 1200 Ser Trp Ser Asn Val Ile Cys Ala Glu Val Leu Gly Ser Met Glu1205 1210 1215 Ile Ala Gly Glu Lys Lys Tyr Gln Tyr Tyr Ser Met Pro SerSer 1220 1225 1230 Leu Leu Arg Ser Leu Ala Gly Gly Arg His Pro Pro ArgVal Gln 1235 1240 1245 Arg Gly Ala Leu Leu Glu Ile Thr Asn Ser Lys ArgGlu Ala Thr 1250 1255 1260 Asn Val Arg Asn Asp Gln Glu Arg Gln Glu ThrGln Ser Ser Ile 1265 1270 1275 Val Val Ser Gly Val Ser Pro Asn Arg GlnAla His Ser Lys Tyr 1280 1285 1290 Gly Gln Phe Leu Leu Val Pro Ser AsnLeu Lys Arg Val Pro Phe 1295 1300 1305 Ser Ala Glu Thr Val Leu Pro LeuSer Arg Pro Ser Val Pro Asp 1310 1315 1320 Val Leu Ala Thr Glu Gln AspIle Gln Thr Glu Val Leu Val His 1325 1330 1335 Leu Thr Gly Gln Thr ProVal Val Ser Asp Trp Ala Ser Val Asp 1340 1345 1350 Glu Pro Lys Glu LysHis Glu Pro Ile Ala His Leu Leu Asp Gly 1355 1360 1365 Gln Asp Lys AlaGlu Gln Val Leu Pro Thr Leu Ser Cys Thr Pro 1370 1375 1380 Glu Pro MetThr Met Ser Ser Pro Leu Ser Gln Ala Lys Ile Met 1385 1390 1395 Gln ThrGly Gly Gly Tyr Val Asn Trp Ala Phe Ser Glu Gly Asp 1400 1405 1410 GluThr Gly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr Cys Leu 1415 1420 1425Pro Ser Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln 1430 14351440 Lys Gln Ala Gln Asp Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser 14451450 1455 Ala Gln Ser Ser Glu Cys Ser Glu Val Gly Pro Trp Leu Gln Pro1460 1465 1470 Asn Thr Ser Phe Trp Ile Asn Pro Leu Arg Arg Tyr Arg ProPhe 1475 1480 1485 Ala Arg Ser His Ser Phe Arg Phe His Lys Glu Glu LysLeu Met 1490 1495 1500 Lys Ile Cys Lys Ile Lys Asn Leu Ser Gly Ser SerGlu Ile Gly 1505 1510 1515 Gln Gly Ala Trp Val Lys Ala Lys Met Leu ThrLys Asp Arg Arg 1520 1525 1530 Leu Ser Lys Lys Lys Lys Asn Thr Gln GlyLeu Gln Val Pro Ile 1535 1540 1545 Ile Thr Val Asn Ala Cys Ser Gln SerAsp Gln Leu Asn Pro Glu 1550 1555 1560 Pro Gly Glu Asn Ser Ile Ser GluGlu Glu Tyr Ser Lys Asn Trp 1565 1570 1575 Phe Thr Val Ser Lys Phe SerHis Thr Gly Val Glu Pro Tyr Ile 1580 1585 1590 His Gln Lys Met Lys ThrLys Glu Ile Gly Gln Cys Ala Ile Gln 1595 1600 1605 Ile Ser Asp Tyr LeuLys Gln Ser Gln Glu Asp Leu Ser Lys Asn 1610 1615 1620 Ser Leu Trp AsnSer Arg Ser Thr Asn Leu Asn Arg Asn Ser Leu 1625 1630 1635 Leu Lys SerSer Ile Gly Val Asp Lys Ile Ser Ala Ser Leu Lys 1640 1645 1650 Ser ProGln Glu Pro His His His Tyr Ser Ala Ile Glu Arg Asn 1655 1660 1665 AsnLeu Met Arg Leu Ser Gln Thr Ile Pro Phe Thr Pro Val Gln 1670 1675 1680Leu Phe Ala Gly Glu Glu Ile Thr Val Tyr Arg Leu Glu Glu Ser 1685 16901695 Ser Pro Leu Asn Leu Asp Lys Ser Met Ser Ser Trp Ser Gln Arg 17001705 1710 Gly Arg Ala Ala Met Ile Gln Val Leu Ser Arg Glu Glu Met Asp1715 1720 1725 Gly Gly Leu Arg Lys Ala Met Arg Val Val Ser Thr Trp SerGlu 1730 1735 1740 Asp Asp Ile Leu Lys Pro Gly Gln Val Phe Ile Val LysSer Phe 1745 1750 1755 Leu Pro Glu Val Val Arg Thr Trp His Lys Ile PheGln Glu Ser 1760 1765 1770 Thr Val Leu His Leu Cys Leu Arg Glu Ile GlnGln Gln Arg Ala 1775 1780 1785 Ala Gln Lys Leu Ile Tyr Thr Phe Asn GlnVal Lys Pro Gln Thr 1790 1795 1800 Ile Pro Tyr Thr Pro Arg Phe Leu GluVal Phe Leu Ile Tyr Cys 1805 1810 1815 His Ser Ala Asn Gln Trp Leu ThrIle Glu Lys Tyr Met Thr Gly 1820 1825 1830 Glu Phe Arg Lys Tyr Asn AsnAsn Asn Gly Asp Glu Ile Thr Pro 1835 1840 1845 Thr Asn Thr Leu Glu GluLeu Met Leu Ala Phe Ser His Trp Thr 1850 1855 1860 Tyr Glu Tyr Thr ArgGly Glu Leu Leu Val Leu Asp Leu Gln Gly 1865 1870 1875 Val Gly Glu AsnLeu Thr Asp Pro Ser Val Ile Lys Pro Glu Val 1880 1885 1890 Lys Gln SerArg Gly Met Val Phe Gly Pro Ala Asn Leu Gly Glu 1895 1900 1905 Asp AlaIle Arg Asn Phe Ile Ala Lys His His Cys Asn Ser Cys 1910 1915 1920 CysArg Lys Leu Lys Leu Pro Asp Leu Lys Arg Asn Asp Tyr Ser 1925 1930 1935Pro Glu Arg Ile Asn Ser Thr Phe Gly Leu Glu Ile Lys Ile Glu 1940 19451950 Ser Ala Glu Glu Pro Pro Ala Arg Glu Thr Gly Arg Asn Ser Pro 19551960 1965 Glu Asp Asp Met Gln Leu 1970 9 1864 PRT Homo sapiens 9 Met SerGln Lys Ser Trp Ile Glu Ser Thr Leu Thr Lys Arg Glu Cys 1 5 10 15 ValTyr Ile Ile Pro Ser Ser Lys Asp Pro His Arg Cys Leu Pro Gly 20 25 30 CysGln Ile Cys Gln Gln Leu Val Arg Cys Phe Cys Gly Arg Leu Val 35 40 45 LysGln His Ala Cys Phe Thr Ala Ser Leu Ala Met Lys Tyr Ser Asp 50 55 60 ValLys Leu Gly Asp His Phe Asn Gln Ala Ile Glu Glu Trp Ser Val 65 70 75 80Glu Lys His Thr Glu Gln Ser Pro Thr Asp Ala Tyr Gly Val Ile Asn 85 90 95Phe Gln Gly Gly Ser His Ser Tyr Arg Ala Lys Tyr Val Arg Leu Ser 100 105110 Tyr Asp Thr Lys Pro Glu Val Ile Leu Gln Leu Leu Leu Lys Glu Trp 115120 125 Gln Met Glu Leu Pro Lys Leu Val Ile Ser Val His Gly Gly Met Gln130 135 140 Lys Phe Glu Leu His Pro Arg Ile Lys Gln Leu Leu Gly Lys GlyLeu 145 150 155 160 Ile Lys Ala Ala Val Thr Thr Gly Ala Trp Ile Leu ThrGly Gly Val 165 170 175 Asn Thr Gly Val Ala Lys His Val Gly Asp Ala LeuLys Glu His Ala 180 185 190 Ser Arg Ser Ser Arg Lys Ile Cys Thr Ile GlyIle Ala Pro Trp Gly 195 200 205 Val Ile Glu Asn Arg Asn Asp Leu Val GlyArg Asp Val Val Ala Pro 210 215 220 Tyr Gln Thr Leu Leu Asn Pro Leu SerLys Leu Asn Val Leu Asn Asn 225 230 235 240 Leu His Ser His Phe Ile LeuVal Asp Asp Gly Thr Val Gly Lys Tyr 245 250 255 Gly Ala Glu Val Arg LeuArg Arg Glu Leu Glu Lys Thr Ile Asn Gln 260 265 270 Gln Arg Ile His AlaArg Ile Gly Gln Gly Val Pro Val Val Ala Leu 275 280 285 Ile Phe Glu GlyGly Pro Asn Val Ile Leu Thr Val Leu Glu Tyr Leu 290 295 300 Gln Glu SerPro Pro Val Pro Val Val Val Cys Glu Gly Thr Gly Arg 305 310 315 320 AlaAla Asp Leu Leu Ala Tyr Ile His Lys Gln Thr Glu Glu Gly Gly 325 330 335Asn Leu Pro Asp Ala Ala Glu Pro Asp Ile Ile Ser Thr Ile Lys Lys 340 345350 Thr Phe Asn Phe Gly Gln Asn Glu Ala Leu His Leu Phe Gln Thr Leu 355360 365 Met Glu Cys Met Lys Arg Lys Glu Leu Ile Thr Val Phe His Ile Gly370 375 380 Ser Asp Glu His Gln Asp Ile Asp Val Ala Ile Leu Thr Ala LeuLeu 385 390 395 400 Lys Gly Thr Asn Ala Ser Ala Phe Asp Gln Leu Ile LeuThr Leu Ala 405 410 415 Trp Asp Arg Val Asp Ile Ala Lys Asn His Val PheVal Tyr Gly Gln 420 425 430 Gln Trp Leu Val Gly Ser Leu Glu Gln Ala MetLeu Asp Ala Leu Val 435 440 445 Met Asp Arg Val Ala Phe Val Lys Leu LeuIle Glu Asn Gly Val Ser 450 455 460 Met His Lys Phe Leu Thr Ile Pro ArgLeu Glu Glu Leu Tyr Asn Thr 465 470 475 480 Lys Gln Gly Pro Thr Asn ProMet Leu Phe His Leu Val Arg Asp Val 485 490 495 Lys Gln Gly Asn Leu ProPro Gly Tyr Lys Ile Thr Leu Ile Asp Ile 500 505 510 Gly Leu Val Ile GluTyr Leu Met Gly Gly Thr Tyr Arg Cys Thr Tyr 515 520 525 Thr Arg Lys ArgPhe Arg Leu Ile Tyr Asn Ser Leu Gly Gly Asn Asn 530 535 540 Arg Arg SerGly Arg Asn Thr Ser Ser Ser Thr Pro Gln Leu Arg Lys 545 550 555 560 SerHis Glu Ser Phe Gly Asn Arg Ala Asp Lys Lys Glu Lys Met Arg 565 570 575His Asn His Phe Ile Lys Thr Ala Gln Pro Tyr Arg Pro Lys Ile Asp 580 585590 Thr Val Met Glu Glu Gly Lys Lys Lys Arg Thr Lys Asp Glu Ile Val 595600 605 Asp Ile Asp Asp Pro Glu Thr Lys Arg Phe Pro Tyr Pro Leu Asn Glu610 615 620 Leu Leu Ile Trp Ala Cys Leu Met Lys Arg Gln Val Met Ala ArgPhe 625 630 635 640 Leu Trp Gln His Gly Glu Glu Ser Met Ala Lys Ala LeuVal Ala Cys 645 650 655 Lys Ile Tyr Arg Ser Met Ala Tyr Glu Ala Lys GlnSer Asp Leu Val 660 665 670 Asp Asp Thr Ser Glu Glu Leu Lys Gln Tyr SerAsn Asp Phe Gly Gln 675 680 685 Leu Ala Val Glu Leu Leu Glu Gln Ser PheArg Gln Asp Glu Thr Met 690 695 700 Ala Met Lys Leu Leu Thr Tyr Glu LeuLys Asn Trp Ser Asn Ser Thr 705 710 715 720 Cys Leu Lys Leu Ala Val SerSer Arg Leu Arg Pro Phe Val Ala His 725 730 735 Thr Cys Thr Gln Met LeuLeu Ser Asp Met Trp Met Gly Arg Leu Asn 740 745 750 Met Arg Lys Asn SerTrp Tyr Lys Val Ile Leu Ser Ile Leu Val Pro 755 760 765 Pro Ala Ile LeuLeu Leu Glu Tyr Lys Thr Lys Ala Glu Met Ser His 770 775 780 Ile Pro GlnSer Gln Asp Ala His Gln Met Thr Met Asp Asp Ser Glu 785 790 795 800 AsnAsn Phe Gln Asn Ile Thr Glu Glu Ile Pro Met Glu Val Phe Lys 805 810 815Glu Val Arg Ile Leu Asp Ser Asn Glu Gly Lys Asn Glu Met Glu Ile 820 825830 Gln Met Lys Ser Lys Lys Leu Pro Ile Thr Arg Lys Phe Tyr Ala Phe 835840 845 Tyr His Ala Pro Ile Val Lys Phe Trp Phe Asn Thr Leu Ala Tyr Leu850 855 860 Gly Phe Leu Met Leu Tyr Thr Phe Val Val Leu Val Gln Met GluGln 865 870 875 880 Leu Pro Ser Val Gln Glu Trp Ile Val Ile Ala Tyr IlePhe Thr Tyr 885 890 895 Ala Ile Glu Lys Val Arg Glu Ile Phe Met Ser GluAla Gly Lys Val 900 905 910 Asn Gln Lys Ile Lys Val Trp Phe Ser Asp TyrPhe Asn Ile Ser Asp 915 920 925 Thr Ile Ala Ile Ile Ser Phe Phe Ile GlyPhe Gly Leu Arg Phe Gly 930 935 940 Ala Lys Trp Asn Phe Ala Asn Ala TyrAsp Asn His Val Phe Val Ala 945 950 955 960 Gly Arg Leu Ile Tyr Cys LeuAsn Ile Ile Phe Trp Tyr Val Arg Leu 965 970 975 Leu Asp Phe Leu Ala ValAsn Gln Gln Ala Gly Pro Tyr Val Met Met 980 985 990 Ile Gly Lys Met ValAla Asn Met Phe Tyr Ile Val Val Ile Met Ala 995 1000 1005 Leu Val LeuLeu Ser Phe Gly Val Pro Arg Lys Ala Ile Leu Tyr 1010 1015 1020 Pro HisGlu Ala Pro Ser Trp Thr Leu Ala Lys Asp Ile Val Phe 1025 1030 1035 HisPro Tyr Trp Met Ile Phe Gly Glu Val Tyr Ala Tyr Glu Ile 1040 1045 1050Asp Val Cys Ala Asn Asp Ser Val Ile Pro Gln Ile Cys Gly Pro 1055 10601065 Gly Thr Trp Leu Thr Pro Phe Leu Gln Ala Val Tyr Leu Phe Val 10701075 1080 Gln Tyr Ile Ile Met Val Asn Leu Leu Ile Ala Phe Phe Asn Asn1085 1090 1095 Val Tyr Leu Gln Val Lys Ala Ile Ser Asn Ile Val Trp LysTyr 1100 1105 1110 Gln Arg Tyr His Phe Ile Met Ala Tyr His Glu Lys ProVal Leu 1115 1120 1125 Pro Pro Pro Leu Ile Ile Leu Ser His Ile Val SerLeu Phe Cys 1130 1135 1140 Cys Ile Cys Lys Arg Arg Lys Lys Asp Lys ThrSer Asp Gly Pro 1145 1150 1155 Lys Leu Phe Leu Thr Glu Glu Asp Gln LysLys Leu His Asp Phe 1160 1165 1170 Glu Glu Gln Cys Val Glu Met Tyr PheAsn Glu Lys Asp Asp Lys 1175 1180 1185 Phe His Ser Gly Ser Glu Glu ArgIle Arg Val Thr Phe Glu Arg 1190 1195 1200 Val Glu Gln Met Cys Ile GlnIle Lys Glu Val Gly Asp Arg Val 1205 1210 1215 Asn Tyr Ile Lys Arg SerLeu Gln Ser Leu Asp Ser Gln Ile Gly 1220 1225 1230 His Leu Gln Asp LeuSer Ala Leu Thr Val Asp Thr Leu Lys Thr 1235 1240 1245 Leu Thr Ala GlnLys Ala Ser Glu Ala Ser Lys Val His Asn Glu 1250 1255 1260 Ile Thr ArgGlu Leu Ser Ile Ser Lys His Leu Ala Gln Asn Leu 1265 1270 1275 Ile AspAsp Gly Pro Val Arg Pro Ser Val Trp Lys Lys His Gly 1280 1285 1290 ValVal Asn Thr Leu Ser Ser Ser Leu Pro Gln Gly Asp Leu Glu 1295 1300 1305Ser Asn Asn Pro Phe His Cys Asn Ile Leu Met Lys Asp Asp Lys 1310 13151320 Asp Pro Gln Cys Asn Ile Phe Gly Gln Asp Leu Pro Ala Val Pro 13251330 1335 Gln Arg Lys Glu Phe Asn Phe Pro Glu Ala Gly Ser Ser Ser Gly1340 1345 1350 Ala Leu Phe Pro Ser Ala Val Ser Pro Pro Glu Leu Arg GlnArg 1355 1360 1365 Leu His Gly Val Glu Leu Leu Lys Ile Phe Asn Lys AsnGln Lys 1370 1375 1380 Leu Gly Ser Ser Ser Thr Ser Ile Pro His Leu SerSer Pro Pro 1385 1390 1395 Thr Lys Phe Phe Val Ser Thr Pro Ser Gln ProSer Cys Lys Ser 1400 1405 1410 His Leu Glu Thr Gly Thr Lys Asp Gln GluThr Val Cys Ser Lys 1415 1420 1425 Ala Thr Glu Gly Asp Asn Thr Glu PheGly Ala Phe Val Gly His 1430 1435 1440 Arg Asp Ser Met Asp Leu Gln ArgPhe Lys Glu Thr Ser Asn Lys 1445 1450 1455 Ile Lys Ile Leu Ser Asn AsnAsn Thr Ser Glu Asn Thr Leu Lys 1460 1465 1470 Arg Val Ser Ser Leu AlaGly Phe Thr Asp Cys His Arg Thr Ser 1475 1480 1485 Ile Pro Val His SerLys Gln Glu Lys Ile Ser Arg Arg Pro Ser 1490 1495 1500 Thr Glu Asp ThrHis Glu Val Asp Ser Lys Ala Ala Leu Ile Pro 1505 1510 1515 Val Trp LeuGln Asp Arg Pro Ser Asn Arg Glu Met Pro Ser Glu 1520 1525 1530 Glu GlyThr Leu Asn Gly Leu Thr Ser Pro Phe Lys Pro Ala Met 1535 1540 1545 AspThr Asn Tyr Tyr Tyr Ser Ala Val Glu Arg Asn Asn Leu Met 1550 1555 1560Arg Leu Ser Gln Ser Ile Pro Phe Thr Pro Val Pro Pro Arg Gly 1565 15701575 Glu Pro Val Thr Val Tyr Arg Leu Glu Glu Ser Ser Pro Asn Ile 15801585 1590 Leu Asn Asn Ser Met Ser Ser Trp Ser Gln Leu Gly Leu Cys Ala1595 1600 1605 Lys Ile Glu Phe Leu Ser Lys Glu Glu Met Gly Gly Gly LeuArg 1610 1615 1620 Arg Ala Val Lys Val Gln Cys Thr Trp Ser Glu His AspIle Leu 1625 1630 1635 Lys Ser Gly His Leu Tyr Ile Ile Lys Ser Phe LeuPro Glu Val 1640 1645 1650 Val Asn Thr Trp Ser Ser Ile Tyr Lys Glu AspThr Val Leu His 1655 1660 1665 Leu Cys Leu Arg Glu Ile Gln Gln Gln ArgAla Ala Gln Lys Leu 1670 1675 1680 Thr Phe Ala Phe Asn Gln Met Lys ProLys Ser Ile Pro Tyr Ser 1685 1690 1695 Pro Arg Phe Leu Glu Val Phe LeuLeu Tyr Cys His Ser Ala Gly 1700 1705 1710 Gln Trp Phe Ala Val Glu GluCys Met Thr Gly Glu Phe Arg Lys 1715 1720 1725 Tyr Asn Asn Asn Asn GlyAsp Glu Ile Ile Pro Thr Asn Thr Leu 1730 1735 1740 Glu Glu Ile Met LeuAla Phe Ser His Trp Thr Tyr Glu Tyr Thr 1745 1750 1755 Arg Gly Glu LeuLeu Val Leu Asp Leu Gln Gly Val Gly Glu Asn 1760 1765 1770 Leu Thr AspPro Ser Val Ile Lys Ala Glu Glu Lys Arg Ser Cys 1775 1780 1785 Asp MetVal Phe Gly Pro Ala Asn Leu Gly Glu Asp Ala Ile Lys 1790 1795 1800 AsnPhe Arg Ala Lys His His Cys Asn Ser Cys Cys Arg Lys Leu 1805 1810 1815Lys Leu Pro Asp Leu Lys Arg Asn Asp Tyr Thr Pro Asp Lys Ile 1820 18251830 Ile Phe Pro Gln Asp Glu Pro Ser Asp Leu Asn Leu Gln Pro Gly 18351840 1845 Asn Ser Thr Lys Glu Ser Glu Ser Thr Asn Ser Val Arg Leu Met1850 1855 1860 Leu 10 240 DNA Homo sapiens 10 catggcagac agtttggccagctggctctg gacttgttgg agaaggcatt caagcagaat 60 gagcgcatgg ccatgacgctgttgacgtat gaactcagga actggagcaa ttcgacctgc 120 cttaaactgg ccgtgtcgggaggattacga ccctttgttt cacatacttg tacccagatg 180 ctactgacag acatgtggatggggaggctg aaaatgagga aaaactcttg gttaaaggta 240 11 258 DNA Homo sapiens11 aaatcaagga agaagtcaaa agaacaaaat gtatcagatg accctgagtc tactggcttt 60ctttaccctt acaatgacct gctggtttgg gctgtgctga tgaaaaggca gaagatggct 120atgttcttct ggcagcatgg agaggaggcc acggttaaag ccgtgattgc gtgtatcctc 180taccgggcaa tggcccatga agctaaggag agtcacatgg tggatgatgc ctcagaagag 240ttgaagaatt actcaaag 258 12 336 DNA Homo sapiens 12 aacatgttct atattgtgatcatcatggcc atagtcctgc tgagctttgg agtggcacgc 60 aaggccatcc tttcgccaaaagagccacca tcttggagtc tagctcgaga tattgtattt 120 gagccatact ggatgatatacggagaagtc tatgctggag aaatagatgg tgtgtatggg 180 attttaccgt catgcagaaattgtgccttc atagttaaca agatttgttc aagccagcca 240 tcctgccctc ctggttcttttcttactcca ttcttgcaag ctgtctacct cttcgtgcaa 300 tatatcatca tggtgaacctgttgattgct ttcttc 336 13 174 DNA Homo sapiens 13 gtggtgtgcc tgtaccagactctggataac cccctcagca agctcacaac actcaacagc 60 atgcactcgc acttcatcctgtctgatgat gggaccgtgg gcaagtatgg aaatgaaatg 120 aagctcagaa ggaacctggagaagtacctc tctctgcaga aaatacactg ccgt 174 14 237 DNA Homo sapiens 14cttacttcat cttacagcaa cgtttactta gatatggaat ccatttcaaa taacctgtgg 60aaatacaacc gctatcgcta catcatgacc taccacgaga agccctggct gcccccacct 120ctcatcctgc tgagccacgt gggccttctc ctccgccgcc tgtgctgtca tcgagctcct 180cacgaccaag aagagggtga cgttggatta agtaagttgt tctatgtgag gcaggag 237 15171 DNA Homo sapiens 15 attaccatat ttgatgctga ctctgaagag cagcaagacctggacttagc aatcctaaca 60 gctttgctga agggcacaaa tttatcagcg tcagagcaattaaatctggc aatggcttgg 120 gacagggtgg acattgccaa gaaacatatc ctaatttatgaacaacactg g 171 16 162 DNA Homo sapiens 16 caaggcgtgc cggtcgtggggctggtggtg gaaggcggtc ccaacgtcat cctgtcagtg 60 tgggagactg tcaaggacaaggacccagtg gtggtgtgtg agggcacagg tagggcggtt 120 gacctcctgg ccttcacacacaaacacctg gcagatgaag gg 162 17 111 DNA Homo sapiens 17 taccgaataaccttgattga cattggatta gtagtagaat acctcattgg tagagcatat 60 cgcagcaactacactagaaa acatttcaga gccctctaca acaacctcta c 111 18 135 DNA Homosapiens 18 ggagtgtcca agcatgttgg ggatgccttg aaatcccatt cctctcattccttgagaaaa 60 atctggacag ttggaatccc tccttggggt gtcattgaga accagagagaccttattgga 120 aaagatgtaa gtaga 135 19 138 DNA Homo sapiens 19atagaactct acctcagtaa ggaggatctg aaaaaacttc atgattttga ggagcagtgc 60gtggaaaaat acttccatga gaagatggaa gatgtgaatt gtagttgtga ggaacgaatc 120cgagtgacat caggaagg 138 20 132 DNA Homo sapiens 20 atggcgtatt tggcattcctcatgctgttc acttacaccg tgttggtgga gatgcagccc 60 cagcccagcg tgcaggagtggcttgttagc atttacatct tcaccaatgc tattgaggtg 120 gtcagggagg tg 132 21 132DNA Homo sapiens 21 atgctgcgac ctcaggtgaa agaggagatc atctgcatgattcagaacac tttcaacttt 60 agttttaaac agtacaagca ccttacccaa atactaatggagtgtatggt tcacagggat 120 tgtgtgagta tg 132 22 78 DNA Homo sapiens 22ttacagatta ttataagcat tattttacca cccaccattt tgacactgga atttaaaagc 60aaagctgaga tgtcacat 78 23 144 DNA Homo sapiens 23 tccattttaa ggctgaataatattcccttg ggtatatatg ccatattttg tttatccatc 60 cagaaaatct atttctatgtcagtggtatt gttctgttgg tctggtggca caacaggtat 120 ttagctcaat ttttaatgattgtg 144 24 99 DNA Homo sapiens 24 aagaaaaaga aagaaaggaa aagaaaagaaaaggaaacga aagagaggaa aggagaggag 60 aggaaaggag aggaagaaag agagagagagaaagagaga 99 25 77 DNA Homo sapiens 25 ttctcaatga caccccaagg agggattccaactgtccaga tttttctcaa ggaatgagag 60 gaatgggatt tcaaggc 77 26 20 DNA Homosapiens 26 taatacgact cactataggg 20 27 19 DNA Homo sapiens 27 gtccaagcatgttggggat 19 28 24 DNA Homo sapiens 28 tcttttccaa taaggtctct ctgg 24 2929 DNA Homo sapiens 29 atgattatcc tatctaagtc ccagaaatc 29 30 24 DNA Homosapiens 30 caaggaatga gaggaatggg attt 24 31 24 DNA Homo sapiens 31aaatcccatt cctctcattc cttg 24 32 24 DNA Homo sapiens 32 gtagcctgaaagaagggtat gctg 24 33 24 DNA Homo sapiens 33 cagcataccc ttctttcagg ctac24 34 24 DNA Homo sapiens 34 gaacatgtga catctcagct ttgc 24 35 24 DNAHomo sapiens 35 gcaaagctga gatgtcacat gttc 24 36 21 DNA Homo sapiens 36agggcaggat ggctggcttg a 21 37 21 DNA Homo sapiens 37 tcaagccagccatcctgccc t 21 38 24 DNA Homo sapiens 38 gcaagtagaa tgctttctct tggc 2439 24 DNA Homo sapiens 39 gccaagagaa agcattctac ttgc 24 40 21 DNA Homosapiens 40 aggggagctc attgtcatgg g 21 41 22 DNA Homo sapiens 41cccatgacaa tgagctcccc tc 22 42 28 DNA Homo sapiens 42 ccaacctgtagacagttatt tcttctcc 28 43 28 DNA Homo sapiens 43 ggagaagaaa taactgtctacaggttgg 28 44 27 DNA Homo sapiens 44 ttatagttgc atatcatctt ctgggga 2745 18 PRT Homo sapiens 45 Leu Lys Ile Ile Ile Ser Ile Ile Leu Pro ProThr Ile Leu Thr Leu 1 5 10 15 Glu Phe 46 18 PRT Homo sapiens 46 Ile ValLys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala Phe Leu Met 1 5 10 15 LeuPhe 47 13 PRT Homo sapiens 47 Thr Glu Thr Val Ala Ile Gly Leu Phe SerAla Gly Phe 1 5 10 48 18 PRT Homo sapiens 48 Arg Leu Ile Tyr Cys Ile AspIle Ile Phe Trp Phe Ser Arg Leu Leu 1 5 10 15 Asp Phe 49 18 PRT Homosapiens 49 Met Thr Ala Asn Met Phe Tyr Ile Val Ile Ile Met Ala Ile ValLeu 1 5 10 15 Leu Ser 50 20 PRT Homo sapiens 50 Phe Leu Gln Ala Val TyrLeu Phe Val Gln Tyr Ile Ile Met Val Asn 1 5 10 15 Leu Leu Ile Ala 20 5113 PRT Homo sapiens 51 Ile Ile Leu Ser Lys Ser Gln Lys Ser Trp Ile LysGly 1 5 10 52 13 PRT Homo sapiens 52 Ser Thr Ile Ile Pro Ser Ser Lys AsnPro His Arg Cys 1 5 10 53 13 PRT Homo sapiens 53 Ser Val Glu Lys His ThrThr Lys Ser Pro Thr Asp Thr 1 5 10 54 13 PRT Homo sapiens 54 Ser His SerSer His Ser Leu Arg Lys Ile Trp Thr Val 1 5 10 55 13 PRT Homo sapiens 55Leu Ser Val Trp Glu Thr Val Lys Asp Lys Asp Pro Val 1 5 10 56 13 PRTHomo sapiens 56 Val Val Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu Leu 1 510 57 13 PRT Homo sapiens 57 Asp Leu Leu Ala Phe Thr His Lys His Leu AlaAsp Glu 1 5 10 58 13 PRT Homo sapiens 58 Asn Thr Phe Asn Phe Ser Leu LysGln Ser Lys His Leu 1 5 10 59 13 PRT Homo sapiens 59 Tyr Arg Ser Asn TyrThr Arg Lys His Phe Arg Ala Leu 1 5 10 60 13 PRT Homo sapiens 60 Ile ValLeu His Lys Ser Arg Lys Lys Ser Lys Glu Gln 1 5 10 61 13 PRT Homosapiens 61 His Gly Glu Glu Ala Thr Val Lys Ala Val Ile Ala Cys 1 5 10 6213 PRT Homo sapiens 62 Asp Gln Asn Ala Ser Ser Ser Lys Glu Ser Ala SerVal 1 5 10 63 13 PRT Homo sapiens 63 Ser Lys Glu Ser Ala Ser Val Lys GluTyr Asp Leu Glu 1 5 10 64 13 PRT Homo sapiens 64 Gln His Leu Pro Trp ThrArg Lys Val Tyr Glu Phe Tyr 1 5 10 65 13 PRT Homo sapiens 65 Glu Pro GlyLys Phe Thr Gln Lys Val Lys Val Trp Ile 1 5 10 66 13 PRT Homo sapiens 66Arg Lys Ala Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp 1 5 10 67 13 PRTHomo sapiens 67 Arg Ile Arg Val Thr Ser Glu Arg Val Thr Glu Met Tyr 1 510 68 13 PRT Homo sapiens 68 Ala Leu Thr Val Asp Thr Leu Lys Val Leu SerAla Val 1 5 10 69 13 PRT Homo sapiens 69 Lys Arg Lys His Ser Thr Cys LysLys Leu Pro His Ser 1 5 10 70 13 PRT Homo sapiens 70 Leu Glu Ile Thr AsnSer Lys Arg Glu Ala Thr Asn Val 1 5 10 71 13 PRT Homo sapiens 71 Glu ThrGly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr 1 5 10 72 13 PRT Homosapiens 72 Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln 1 5 10 7313 PRT Homo sapiens 73 Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln SerSer 1 5 10 74 13 PRT Homo sapiens 74 Phe Ala Arg Ser His Ser Phe Arg PheHis Lys Glu Glu 1 5 10 75 13 PRT Homo sapiens 75 Lys Asp Arg Arg Leu SerLys Lys Lys Lys Asn Thr Gln 1 5 10 76 13 PRT Homo sapiens 76 Asp Lys IleSer Ala Ser Leu Lys Ser Pro Gln Glu Pro 1 5 10 77 13 PRT Homo sapiens 77Ser Met Ser Ser Trp Ser Gln Arg Gly Arg Ala Ala Met 1 5 10 78 13 PRTHomo sapiens 78 Gln Thr Ile Pro Tyr Thr Pro Arg Phe Leu Glu Val Phe 1 510 79 13 PRT Homo sapiens 79 Pro Pro Ala Arg Glu Thr Gly Arg Asn Ser ProGlu Asp 1 5 10 80 14 PRT Homo sapiens 80 His Gly Gly Ile Gln Asn Phe ThrMet Pro Ser Lys Phe Lys 1 5 10 81 14 PRT Homo sapiens 81 Ile Gln Asn ThrPhe Asn Phe Ser Leu Lys Gln Ser Lys His 1 5 10 82 14 PRT Homo sapiens 82Leu Leu Lys Gly Thr Asn Leu Ser Ala Ser Glu Gln Leu Asn 1 5 10 83 14 PRTHomo sapiens 83 Arg Ala Tyr Arg Ser Asn Tyr Thr Arg Lys His Phe Arg Ala1 5 10 84 14 PRT Homo sapiens 84 Ser Ser Gly Asn Arg Asn Glu Ser Ala GluSer Thr Leu His 1 5 10 85 14 PRT Homo sapiens 85 Lys Ser Lys Glu Gln AsnVal Ser Asp Asp Pro Glu Ser Thr 1 5 10 86 14 PRT Homo sapiens 86 Ser GluGlu Leu Lys Asn Tyr Ser Lys Gln Phe Gly Gln Leu 1 5 10 87 14 PRT Homosapiens 87 Thr Tyr Glu Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys 1 510 88 14 PRT Homo sapiens 88 Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu LysLeu Ala Val 1 5 10 89 14 PRT Homo sapiens 89 Tyr Tyr Ser Asp Gln Asn AlaSer Ser Ser Lys Glu Ser Ala 1 5 10 90 14 PRT Homo sapiens 90 Ile Ser GluTyr Trp Asn Leu Thr Glu Thr Val Ala Ile Gly 1 5 10 91 14 PRT Homosapiens 91 Lys Met Glu Asp Val Asn Cys Ser Cys Glu Glu Arg Ile Arg 1 510 92 14 PRT Homo sapiens 92 Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser AlaGln Ser Ser 1 5 10 93 14 PRT Homo sapiens 93 Pro Trp Leu Gln Pro Asn ThrSer Phe Trp Ile Asn Pro Leu 1 5 10 94 14 PRT Homo sapiens 94 Ile Cys LysIle Lys Asn Leu Ser Gly Ser Ser Glu Ile Gly 1 5 10 95 14 PRT Homosapiens 95 Gln Gly Val Gly Glu Asn Leu Thr Asp Pro Ser Val Ile Lys 1 510 96 14 PRT Homo sapiens 96 Ser Pro Glu Arg Ile Asn Ser Thr Phe Gly LeuGlu Ile Lys 1 5 10 97 1933 DNA Homo sapiens 97 caagttctct ggtctccacccaaagatgat tatcctatct aagtcccaga aatcctggat 60 taaaggagta tttgacaagagagaatgtag cacaatcata cccagctcaa aaaatcctca 120 cagatgtact ccagtatgccaagtctgcca gaatttaatc aggtgttact gtggccgact 180 gattggagac catgctgggatagattattc ctggaccatc tcagctgcca agggtaaaga 240 aagtgaacaa tggtctgttgaaaagcacac aacgaaaagc ccaacagata cttttggcac 300 gattaatttc caagatggagagcacaccca tcatgccaag tatattagaa cttcttatga 360 tacaaaactg gatcatctgttacatttaat gttgaaagag tggaaaatgg aactgcccaa 420 gcttgtgatc tcagtccatgggggcatcca gaactttact atgccctcta aatttaaaga 480 gattttcagc caaggtttggttaaagctgc agagacaaca ggagcgtgga taataactga 540 aggcatcaat acaggagtgtccaagcatgt tggggatgcc ttgaaatccc attcctctca 600 ttccttgaga aaaatctggacagttggaat ccctccttgg ggtgtcattg agaaccagag 660 agaccttatt ggaaaagatgtggtgtgcct gtaccagact ctggataacc ccctcagcaa 720 gctcacaaca ctcaacagcatgcactcgca cttcatcctg tctgatgatg ggaccgtggg 780 caagtatgga aatgaaatgaagctcagaag gaacctggag aagtacctct ctctgcagaa 840 aatacactgc cgctcaagacaaggcgtgcc ggtcgtgggg ctggtggtgg aaggcggtcc 900 caacgtcatc ctgtcagtgtgggagactgt caaggacaag gacccagtgg tggtgtgtga 960 gggcacaggt agggcggctgacctcctggc cttcacacac aaacacctgg cagatgaagg 1020 gatgctgcga cctcaggtgaaagaggagat catctgcatg attcagaaca ctttcaactt 1080 tagtcttaaa cagtccaagcaccttttcca aattctaatg gagtgtatgg ttcacaggga 1140 ttgtattacc atatttgatgctgactctga agagcagcaa gacctggact tagcaatcct 1200 aacagctttg ctgaagggcacaaatttatc agcgtcagag caattaaatc tggcaatggc 1260 ttgggacagg gtggacattgccaagaaaca tatcctaatt tatgaacaac actggaagcc 1320 tgatgccctg gaacaagcaatgtcagatgc tttagtgatg gatcgggtgg attttgtgaa 1380 gctcttaata gaatatggagtgaacctcca tcgctttctt accatccctc gactggaaga 1440 gctctacaat acaaaacaaggacctactaa tacactcttg catcatctcg tccaagatgt 1500 gaaacaggta acctaattaagaaggcgtga acatgtcttt gtgtattgcc ttagctttgt 1560 gttggaggag tcactgaattttgctttgta aaatgcagat ttggattgtt ttgttaatta 1620 cctatcttct ttaccttgatttgaacaagt tcatcaacaa accacattcg tacacttttt 1680 tccttaattt actttatgttttaaaagact tgatccatca gttaggtgat atttgctaat 1740 aaaaagccat gaatcagagacatgcaatgc ttctaatcag aacttcagct taccccgata 1800 ccgtggtggt gcctatgatgatgataccag ctggaaataa ataactttgt ggttgtgtta 1860 tccttaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920 aaaaaaaaaa aaa 1933 98496 PRT Homo sapiens 98 Met Ile Ile Leu Ser Lys Ser Gln Lys Ser Trp IleLys Gly Val Phe 1 5 10 15 Asp Lys Arg Glu Cys Ser Thr Ile Ile Pro SerSer Lys Asn Pro His 20 25 30 Arg Cys Thr Pro Val Cys Gln Val Cys Gln AsnLeu Ile Arg Cys Tyr 35 40 45 Cys Gly Arg Leu Ile Gly Asp His Ala Gly IleAsp Tyr Ser Trp Thr 50 55 60 Ile Ser Ala Ala Lys Gly Lys Glu Ser Glu GlnTrp Ser Val Glu Lys 65 70 75 80 His Thr Thr Lys Ser Pro Thr Asp Thr PheGly Thr Ile Asn Phe Gln 85 90 95 Asp Gly Glu His Thr His His Ala Lys TyrIle Arg Thr Ser Tyr Asp 100 105 110 Thr Lys Leu Asp His Leu Leu His LeuMet Leu Lys Glu Trp Lys Met 115 120 125 Glu Leu Pro Lys Leu Val Ile SerVal His Gly Gly Ile Gln Asn Phe 130 135 140 Thr Met Pro Ser Lys Phe LysGlu Ile Phe Ser Gln Gly Leu Val Lys 145 150 155 160 Ala Ala Glu Thr ThrGly Ala Trp Ile Ile Thr Glu Gly Ile Asn Thr 165 170 175 Gly Val Ser LysHis Val Gly Asp Ala Leu Lys Ser His Ser Ser His 180 185 190 Ser Leu ArgLys Ile Trp Thr Val Gly Ile Pro Pro Trp Gly Val Ile 195 200 205 Glu AsnGln Arg Asp Leu Ile Gly Lys Asp Val Val Cys Leu Tyr Gln 210 215 220 ThrLeu Asp Asn Pro Leu Ser Lys Leu Thr Thr Leu Asn Ser Met His 225 230 235240 Ser His Phe Ile Leu Ser Asp Asp Gly Thr Val Gly Lys Tyr Gly Asn 245250 255 Glu Met Lys Leu Arg Arg Asn Leu Glu Lys Tyr Leu Ser Leu Gln Lys260 265 270 Ile His Cys Arg Ser Arg Gln Gly Val Pro Val Val Gly Leu ValVal 275 280 285 Glu Gly Gly Pro Asn Val Ile Leu Ser Val Trp Glu Thr ValLys Asp 290 295 300 Lys Asp Pro Val Val Val Cys Glu Gly Thr Gly Arg AlaAla Asp Leu 305 310 315 320 Leu Ala Phe Thr His Lys His Leu Ala Asp GluGly Met Leu Arg Pro 325 330 335 Gln Val Lys Glu Glu Ile Ile Cys Met IleGln Asn Thr Phe Asn Phe 340 345 350 Ser Leu Lys Gln Ser Lys His Leu PheGln Ile Leu Met Glu Cys Met 355 360 365 Val His Arg Asp Cys Ile Thr IlePhe Asp Ala Asp Ser Glu Glu Gln 370 375 380 Gln Asp Leu Asp Leu Ala IleLeu Thr Ala Leu Leu Lys Gly Thr Asn 385 390 395 400 Leu Ser Ala Ser GluGln Leu Asn Leu Ala Met Ala Trp Asp Arg Val 405 410 415 Asp Ile Ala LysLys His Ile Leu Ile Tyr Glu Gln His Trp Lys Pro 420 425 430 Asp Ala LeuGlu Gln Ala Met Ser Asp Ala Leu Val Met Asp Arg Val 435 440 445 Asp PheVal Lys Leu Leu Ile Glu Tyr Gly Val Asn Leu His Arg Phe 450 455 460 LeuThr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr Lys Gln Gly Pro 465 470 475480 Thr Asn Thr Leu Leu His His Leu Val Gln Asp Val Lys Gln Val Thr 485490 495 99 461 PRT Caenorhabditis elegans 99 Lys Phe Ile Phe Asp Leu MetVal Cys Gly Lys Thr Asn Asp Asn Glu 1 5 10 15 Pro Leu Gln Glu Phe IleLeu Gln Ser Pro Ala Pro Ile Glu Thr Ala 20 25 30 Val Lys Leu Ser Ala LeuTyr Arg Asp Met Ser Glu Lys Glu Lys Glu 35 40 45 Arg Ala Lys Asp Leu LeuAsn Val Ala Val Phe Ser Glu Asn Met Ala 50 55 60 Val Glu Leu Leu Gly IleThr Ala Thr Glu Tyr Asn Ala Ala Leu Leu 65 70 75 80 Leu Lys Ala Lys AspAsn Arg Gly Arg Pro Leu Leu Asp Val Leu Ile 85 90 95 Glu Asn Glu Gln LysGlu Val Val Ser Tyr Ala Ser Val Gln Arg Tyr 100 105 110 Leu Thr Glu ValTrp Thr Ala Arg Val Asp Trp Ser Phe Gly Lys Phe 115 120 125 Val Ala PheSer Leu Phe Val Leu Ile Cys Pro Pro Ala Trp Phe Tyr 130 135 140 Phe SerLeu Pro Leu Asp Ser Arg Ile Gly Arg Ala Pro Ile Ile Lys 145 150 155 160Phe Val Cys His Ile Val Ser His Val Tyr Phe Thr Ile Leu Leu Thr 165 170175 Ile Val Val Leu Asn Ile Thr His Lys Met Tyr Glu Val Thr Ser Val 180185 190 Val Pro Asn Pro Val Glu Trp Leu Leu Leu Leu Trp Leu Ser Gly Asn195 200 205 Leu Val Ser Glu Leu Ser Thr Val Gly Gly Gly Ser Gly Leu GlyIle 210 215 220 Val Lys Val Leu Ile Leu Val Leu Ser Ala Met Ala Ile AlaVal His 225 230 235 240 Val Leu Ala Phe Leu Leu Pro Ala Val Phe Leu ThrHis Leu Asp Asn 245 250 255 Asp Glu Lys Leu His Phe Ala Arg Thr Met LeuTyr Leu Lys Asn Gln 260 265 270 Leu Phe Ala Phe Ala Leu Leu Phe Ala PheVal Glu Tyr Leu Asp Phe 275 280 285 Leu Thr Val His His Leu Phe Gly ProTrp Ala Ile Ile Ile Arg Asp 290 295 300 Leu Met Tyr Asp Leu Ala Arg PheLeu Val Ile Leu Met Leu Phe Val 305 310 315 320 Ala Gly Phe Thr Leu HisVal Thr Ser Ile Phe Gln Pro Ala Tyr Gln 325 330 335 Pro Val Asp Glu AspSer Ala Glu Leu Met Arg Leu Ala Ser Pro Ser 340 345 350 Gln Thr Leu GluMet Leu Phe Phe Ser Leu Phe Gly Leu Val Glu Pro 355 360 365 Asp Ser MetPro Pro Leu His Leu Val Pro Asp Phe Ala Lys Ile Ile 370 375 380 Leu LysLeu Leu Phe Gly Ile Tyr Met Met Val Thr Leu Ile Val Leu 385 390 395 400Ile Asn Leu Leu Ile Ala Met Met Ser Asp Thr Tyr Gln Arg Ile Gln 405 410415 Ala Gln Ser Asp Lys Glu Trp Lys Phe Gly Arg Ala Ile Leu Ile Arg 420425 430 Gln Met Asn Lys Lys Ser Ala Thr Pro Ser Pro Ile Asn Met Leu Thr435 440 445 Lys Leu Ile Ile Val Leu Arg Val Ala Trp Arg Asn Arg 450 455460 100 476 PRT Drosophila melanogaster 100 Arg Phe Val Tyr Asn Leu MetVal Val Ser Lys Asn His Asn Asn Lys 1 5 10 15 Pro Ile Gln Glu Phe ValLeu Val Ser Pro Ala Pro Val Asp Thr Ala 20 25 30 Ala Lys Leu Ser Asn IleTyr Ile Val Leu Ser Thr Lys Glu Lys Glu 35 40 45 Arg Ala Lys Asp Leu ValAla Ala Gly Lys Gln Cys Glu Ala Met Ala 50 55 60 Thr Glu Leu Leu Ala LeuAla Ala Gly Ser Asp Ser Ala Gly Lys Ile 65 70 75 80 Leu Gln Ala Thr AspLys Arg Asn Val Glu Phe Leu Asp Val Leu Ile 85 90 95 Glu Asn Glu Gln LysGlu Val Ile Ala His Thr Val Val Gln Arg Tyr 100 105 110 Leu Gln Glu LeuTrp His Gly Ser Leu Thr Trp Ala Ser Trp Lys Ile 115 120 125 Leu Leu LeuLeu Val Ala Phe Ile Val Cys Pro Pro Val Trp Ile Gly 130 135 140 Phe ThrPhe Pro Met Gly His Lys Phe Asn Lys Val Pro Ile Ile Lys 145 150 155 160Phe Met Ser Tyr Leu Thr Ser His Ile Tyr Leu Met Ile His Leu Ser 165 170175 Ile Val Gly Ile Thr Pro Ile Tyr Pro Val Leu Arg Leu Ser Leu Val 180185 190 Pro Tyr Trp Tyr Glu Val Gly Leu Leu Ile Trp Leu Ser Gly Leu Leu195 200 205 Leu Phe Glu Leu Thr Asn Pro Ser Asp Lys Ser Gly Leu Gly SerIle 210 215 220 Lys Val Leu Val Leu Leu Leu Gly Met Ala Gly Val Gly ValHis Val 225 230 235 240 Ser Ala Phe Leu Phe Val Ser Lys Glu Tyr Trp ProThr Leu Val Tyr 245 250 255 Cys Arg Asn Gln Cys Phe Ala Leu Ala Phe LeuLeu Ala Cys Val Gln 260 265 270 Ile Leu Asp Phe Leu Ser Phe His His LeuPhe Gly Pro Trp Ala Ile 275 280 285 Ile Ile Gly Asp Leu Leu Lys Asp LeuAla Arg Phe Leu Ala Val Leu 290 295 300 Ala Ile Phe Val Phe Gly Phe SerMet His Ile Val Ala Leu Asn Gln 305 310 315 320 Ser Phe Ala Asn Phe SerPro Glu Asp Leu Arg Ser Phe Glu Lys Lys 325 330 335 Asn Arg Asn Arg GlyTyr Phe Ser Asp Val Arg Met His Pro Ile Asn 340 345 350 Ser Phe Glu LeuLeu Phe Phe Ala Val Phe Gly Gln Thr Thr Thr Glu 355 360 365 Gln Thr GlnVal Asp Lys Ile Lys Asn Val Ala Thr Pro Thr Gln Pro 370 375 380 Tyr TrpVal Glu Tyr Leu Phe Lys Ile Val Phe Gly Ile Tyr Met Leu 385 390 395 400Val Ser Val Val Val Leu Ile Asn Leu Leu Ile Ala Met Met Ser Asp 405 410415 Thr Tyr Gln Arg Ile Gln Val Val Leu Leu Asn Ala Leu Leu Ser Asn 420425 430 Ser Thr Leu Phe Ile Asn Ser Tyr Phe Asn His Lys Tyr Ile Asn Phe435 440 445 Ile Leu His Cys Val Leu Ile Ile Leu Tyr Phe Ser Ile Arg SerLys 450 455 460 Phe Thr Tyr Glu Asp Asp Leu Tyr Phe Leu Asp Ile 465 470475 101 862 PRT Mus musculus 101 Met Leu Gly Ser Asn Thr Phe Lys Asn MetGln Arg Arg His Thr Thr 1 5 10 15 Leu Arg Glu Lys Gly Arg Arg Gln AlaIle Arg Gly Pro Ala Tyr Met 20 25 30 Phe Asn Glu Lys Gly Thr Ser Leu ThrPro Glu Glu Glu Arg Phe Leu 35 40 45 Asp Ser Ala Glu Tyr Gly Asn Ile ProVal Val Arg Lys Met Leu Glu 50 55 60 Glu Ser Lys Thr Leu Asn Phe Asn CysVal Asp Tyr Met Gly Gln Asn 65 70 75 80 Ala Leu Gln Leu Ala Val Gly AsnGlu His Leu Glu Val Thr Glu Leu 85 90 95 Leu Leu Lys Lys Glu Asn Leu AlaArg Val Gly Asp Ala Leu Leu Leu 100 105 110 Ala Ile Ser Lys Gly Tyr ValArg Ile Val Glu Ala Ile Leu Ser His 115 120 125 Pro Ala Phe Ala Gln GlyGln Arg Leu Thr Leu Ser Pro Leu Glu Gln 130 135 140 Glu Leu Arg Asp AspAsp Phe Tyr Ala Tyr Asp Glu Asp Gly Thr Arg 145 150 155 160 Phe Ser HisAsp Ile Thr Pro Ile Ile Leu Ala Ala His Cys Gln Glu 165 170 175 Tyr GluIle Val His Ile Leu Leu Leu Lys Gly Ala Arg Ile Glu Arg 180 185 190 ProHis Asp Tyr Phe Cys Lys Cys Asn Glu Cys Thr Glu Lys Gln Arg 195 200 205Lys Asp Ser Phe Ser His Ser Arg Ser Arg Met Asn Ala Tyr Lys Gly 210 215220 Leu Ala Ser Ala Ala Tyr Leu Ser Leu Ser Ser Glu Asp Pro Val Leu 225230 235 240 Thr Ala Leu Glu Leu Ser Asn Glu Leu Ala Arg Leu Ala Asn IleGlu 245 250 255 Thr Glu Phe Lys Asn Asp Tyr Arg Lys Leu Ser Met Gln CysLys Asp 260 265 270 Phe Val Val Gly Val Leu Asp Leu Cys Arg Asp Thr GluGlu Val Glu 275 280 285 Ala Ile Leu Asn Gly Asp Val Asn Leu Gln Val TrpSer Asp His His 290 295 300 Arg Pro Ser Leu Ser Arg Ile Lys Leu Ala IleLys Tyr Glu Val Lys 305 310 315 320 Lys Phe Val Ala His Pro Asn Cys GlnGln Gln Leu Leu Thr Met Trp 325 330 335 Tyr Glu Asn Leu Ser Gly Leu ArgGln Gln Ser Ile Ala Val Lys Phe 340 345 350 Leu Ala Val Phe Gly Val SerIle Gly Leu Pro Phe Leu Ala Ile Ala 355 360 365 Tyr Trp Ile Ala Pro CysSer Lys Leu Gly Gln Thr Leu Arg Ser Pro 370 375 380 Phe Met Lys Phe ValAla His Ala Val Ser Phe Thr Ile Phe Leu Gly 385 390 395 400 Leu Leu ValVal Asn Ala Ser Asp Arg Phe Glu Gly Val Lys Thr Leu 405 410 415 Pro AsnGlu Thr Phe Thr Asp Tyr Pro Lys Gln Ile Phe Arg Val Lys 420 425 430 ThrThr Gln Phe Ser Trp Thr Glu Met Leu Ile Met Lys Trp Val Leu 435 440 445Gly Met Ile Trp Ser Glu Cys Lys Glu Ile Trp Glu Glu Gly Pro Arg 450 455460 Glu Tyr Val Leu His Leu Trp Asn Leu Leu Asp Phe Gly Met Leu Ser 465470 475 480 Ile Phe Val Ala Ser Phe Thr Ala Arg Phe Met Ala Phe Leu LysAla 485 490 495 Ser Glu Ala Gln Leu Tyr Val Asp Gln Tyr Val Gln Asp ValThr Leu 500 505 510 His Asn Val Ser Leu Pro Pro Glu Val Ala Tyr Phe ThrTyr Ala Arg 515 520 525 Asp Lys Trp Trp Pro Ser Asp Pro Gln Ile Ile SerGlu Gly Leu Tyr 530 535 540 Ala Ile Ala Val Val Leu Ser Phe Ser Arg IleAla Tyr Ile Leu Pro 545 550 555 560 Ala Asn Glu Ser Phe Gly Pro Leu GlnIle Ser Leu Gly Arg Thr Val 565 570 575 Lys Asp Ile Phe Lys Phe Met ValIle Phe Ile Met Val Phe Val Ala 580 585 590 Phe Met Ile Gly Met Phe AsnLeu Tyr Ser Tyr Tyr Arg Gly Ala Lys 595 600 605 Tyr Asn Pro Ala Phe ThrThr Val Glu Glu Ser Phe Lys Thr Leu Phe 610 615 620 Trp Ser Ile Phe GlyLeu Ser Glu Val Ile Ser Val Val Leu Lys Tyr 625 630 635 640 Asp His LysPhe Ile Glu Asn Ile Gly Tyr Val Leu Tyr Gly Val Tyr 645 650 655 Asn ValThr Met Val Val Val Leu Leu Asn Met Leu Ile Ala Met Ile 660 665 670 AsnAsn Ser Tyr Gln Glu Ile Glu Glu Asp Ala Asp Val Glu Trp Lys 675 680 685Phe Ala Arg Ala Lys Leu Trp Leu Ser Tyr Phe Asp Glu Gly Arg Thr 690 695700 Leu Pro Ala Pro Phe Asn Leu Val Pro Ser Pro Lys Ser Phe Tyr Tyr 705710 715 720 Leu Ile Met Arg Ile Lys Met Cys Leu Ile Glu Leu Cys Gln SerLys 725 730 735 Ala Lys Arg Cys Glu Asn Asp Leu Glu Met Gly Met Leu AsnSer Lys 740 745 750 Phe Arg Lys Thr Arg Tyr Gln Ala Gly Met Arg Asn SerGlu Asn Leu 755 760 765 Thr Ala Asn Ser Thr Phe Ser Lys Pro Thr Arg TyrGln Lys Ile Met 770 775 780 Lys Arg Leu Ile Lys Arg Tyr Val Leu Lys AlaGln Val Asp Arg Glu 785 790 795 800 Asn Asp Glu Val Asn Glu Gly Glu LeuLys Glu Ile Lys Gln Asp Ile 805 810 815 Ser Ser Leu Arg Tyr Glu Leu LeuGlu Glu Lys Ser Gln Ala Thr Gly 820 825 830 Glu Leu Ala Asp Leu Ile GlnGln Leu Ser Glu Lys Phe Gly Lys Asn 835 840 845 Leu Asn Lys Asp His LeuArg Val Asn Gln Gly Lys Asp Ile 850 855 860 102 930 PRT Mus musculus 102Met Ser Gln Ser Pro Arg Phe Val Thr Arg Arg Gly Gly Ser Leu Lys 1 5 1015 Ala Ala Pro Gly Ala Gly Thr Arg Arg Asn Glu Ser Gln Asp Tyr Leu 20 2530 Leu Met Asp Glu Leu Gly Asp Asp Gly Tyr Pro Gln Leu Pro Leu Pro 35 4045 Pro Tyr Gly Tyr Tyr Pro Ser Phe Arg Gly Asn Glu Asn Arg Leu Thr 50 5560 His Arg Arg Gln Thr Ile Leu Arg Glu Lys Gly Arg Arg Leu Ala Asn 65 7075 80 Arg Gly Pro Ala Tyr Met Phe Asn Asp His Ser Thr Ser Leu Ser Ile 8590 95 Glu Glu Glu Arg Phe Leu Asp Ala Val Glu Tyr Gly Asn Ile Pro Val100 105 110 Val Trp Lys Met Leu Glu Glu Cys His Ser Leu Asn Val Asn CysVal 115 120 125 Asp Tyr Met Gly Gln Asn Ala Leu Gln Leu Ala Val Ala AsnGlu His 130 135 140 Leu Glu Ile Thr Glu Leu Leu Leu Lys Lys Glu Asn LeuSer Arg Val 145 150 155 160 Gly Asp Ala Leu Leu Leu Ala Ile Ser Lys GlyTyr Val Arg Ile Val 165 170 175 Glu Ala Ile Leu Asn His Pro Ser Phe AlaGlu Gly Lys Arg Leu Ala 180 185 190 Thr Ser Pro Ser Gln Ser Glu Leu GlnGln Asp Asp Phe Tyr Ala Tyr 195 200 205 Asp Glu Asp Gly Thr Arg Phe SerHis Asp Val Thr Pro Ile Ile Leu 210 215 220 Ala Ala His Cys Gln Glu TyrGlu Ile Val His Thr Leu Leu Arg Lys 225 230 235 240 Gly Ala Arg Ile GluArg Pro His Asp Tyr Phe Cys Lys Cys Thr Glu 245 250 255 Cys Ser Gln LysGln Lys His Asp Ser Phe Ser His Ser Arg Ser Arg 260 265 270 Ile Asn AlaTyr Lys Gly Leu Ala Ser Pro Ala Tyr Leu Ser Leu Ser 275 280 285 Ser GluAsp Pro Val Met Thr Ala Leu Glu Leu Ser Asn Glu Leu Ala 290 295 300 ValLeu Ala Asn Ile Glu Lys Glu Phe Lys Asn Asp Tyr Arg Lys Leu 305 310 315320 Ser Met Gln Cys Lys Asp Phe Val Val Gly Leu Leu Asp Leu Cys Arg 325330 335 Asn Thr Glu Glu Val Glu Ala Ile Leu Asn Gly Asp Ala Glu Thr Arg340 345 350 Gln Pro Gly Asp Phe Gly Arg Pro Asn Leu Ser Arg Leu Lys LeuAla 355 360 365 Ile Lys Asp Glu Val Lys Lys Phe Val Ala His Pro Asn CysGln Gln 370 375 380 Gln Leu Leu Ser Ile Trp Tyr Glu Asn Leu Ser Gly LeuArg Gln Gln 385 390 395 400 Thr Met Ala Val Lys Phe Leu Val Val Leu AlaVal Ala Ile Gly Leu 405 410 415 Pro Phe Leu Ala Leu Ile Tyr Trp Cys AlaPro Cys Ser Lys Met Gly 420 425 430 Lys Ile Leu Pro Arg Pro Phe Met LysPhe Val Ala His Ala Ala Ser 435 440 445 Phe Thr Ile Phe Leu Gly Leu LeuVal Met Asn Ala Ala Asp Arg Phe 450 455 460 Glu Gly Thr Lys Leu Leu ProAsn Glu Thr Ser Thr Asp Asn Ala Arg 465 470 475 480 Gln Leu Phe Arg MetLys Thr Ser Cys Phe Ser Trp Met Glu Met Leu 485 490 495 Ile Ile Ser TrpVal Ile Gly Met Ile Trp Ala Glu Cys Lys Glu Ile 500 505 510 Trp Thr GlnGly Pro Lys Glu Tyr Leu Phe Glu Leu Trp Asn Met Leu 515 520 525 Asp PheGly Met Leu Ala Ile Phe Ala Ala Ser Phe Ile Ala Arg Phe 530 535 540 MetAla Phe Trp His Ala Ser Lys Ala Gln Ser Ile Ile Asp Ala Asn 545 550 555560 Asp Thr Leu Lys Asp Leu Thr Lys Val Thr Leu Gly Asp Asn Val Lys 565570 575 Tyr Tyr Asn Leu Ala Arg Ile Lys Trp Asp Pro Thr Asp Pro Gln Ile580 585 590 Ile Ser Glu Gly Leu Tyr Ala Ile Ala Val Val Leu Ser Phe SerArg 595 600 605 Ile Ala Tyr Ile Leu Pro Ala Asn Glu Ser Phe Gly Pro LeuGln Ile 610 615 620 Ser Leu Gly Arg Thr Val Lys Asp Ile Phe Lys Phe MetVal Ile Phe 625 630 635 640 Ile Met Val Phe Val Ala Phe Met Ile Gly MetPhe Asn Leu Tyr Ser 645 650 655 Tyr Tyr Ile Gly Ala Lys Gln Asn Glu AlaPhe Thr Thr Val Glu Glu 660 665 670 Ser Phe Lys Thr Leu Phe Trp Ala IlePhe Gly Leu Ser Glu Val Lys 675 680 685 Ser Val Val Ile Asn Tyr Asn HisLys Phe Ile Glu Asn Ile Gly Tyr 690 695 700 Val Leu Tyr Gly Val Tyr AsnVal Thr Met Val Ile Val Leu Leu Asn 705 710 715 720 Met Leu Ile Ala MetIle Asn Ser Ser Phe Gln Glu Ile Glu Asp Asp 725 730 735 Ala Asp Val GluTrp Lys Phe Ala Arg Ala Lys Leu Trp Phe Ser Tyr 740 745 750 Phe Glu GluGly Arg Thr Leu Pro Val Pro Phe Asn Leu Val Pro Ser 755 760 765 Pro LysSer Leu Leu Tyr Leu Leu Leu Lys Phe Lys Lys Trp Met Cys 770 775 780 GluLeu Ile Gln Gly Gln Lys Gln Gly Phe Gln Glu Asp Ala Glu Met 785 790 795800 Asn Lys Arg Asn Glu Glu Lys Lys Phe Gly Ile Ser Gly Ser His Glu 805810 815 Asp Leu Ser Lys Phe Ser Leu Asp Lys Asn Gln Leu Ala His Asn Lys820 825 830 Gln Ser Ser Thr Arg Ser Ser Glu Asp Tyr His Leu Asn Ser PheSer 835 840 845 Asn Pro Pro Arg Gln Tyr Gln Lys Ile Met Lys Arg Leu IleLys Arg 850 855 860 Tyr Val Leu Gln Ala Gln Ile Asp Lys Glu Ser Asp GluVal Asn Glu 865 870 875 880 Gly Glu Leu Lys Glu Ile Lys Gln Asp Ile SerSer Leu Arg Tyr Glu 885 890 895 Leu Leu Glu Glu Lys Ser Gln Asn Ser GluAsp Leu Ala Glu Leu Ile 900 905 910 Arg Lys Leu Gly Glu Arg Leu Ser LeuGlu Pro Lys Leu Glu Glu Ser 915 920 925 Arg Arg 930 103 836 PRT Musmusculus 103 Met Arg Asp Lys Gly Arg Arg Gln Ala Val Arg Gly Pro Ala PheMet 1 5 10 15 Phe Gly Ala Arg Gly Pro Ser Leu Thr Ala Glu Glu Glu ArgPhe Leu 20 25 30 Asp Ala Ala Glu Tyr Gly Asn Ile Pro Val Val Arg Lys MetLeu Glu 35 40 45 Glu Ser Arg Thr Leu Asn Val Asn Cys Val Asp Tyr Met GlyGln Asn 50 55 60 Ala Leu Gln Leu Ala Val Gly Asn Glu His Leu Glu Val ThrGlu Leu 65 70 75 80 Leu Leu Lys Lys Glu Asn Leu Ala Arg Ile Gly Asp AlaLeu Leu Leu 85 90 95 Ala Ile Ser Lys Gly Tyr Val Arg Ile Val Glu Ala IleLeu Gly His 100 105 110 Pro Gly Phe Ala Ala Ser Arg Arg Leu Thr Leu SerPro Cys Glu Gln 115 120 125 Glu Leu Arg Asp Asp Asp Phe Tyr Ala Tyr AspGlu Asp Gly Thr Arg 130 135 140 Phe Ser Pro Asp Ile Thr Pro Ile Ile LeuAla Ala His Cys His Lys 145 150 155 160 Tyr Glu Val Val His Leu Leu LeuLeu Lys Gly Ala Arg Ile Glu Arg 165 170 175 Ala His Asp Tyr Phe Cys ArgCys Ser Asp Cys Ala Glu Lys Gln Arg 180 185 190 Leu Asp Ala Phe Ser HisSer Arg Ser Arg Ile Asn Ala Tyr Lys Gly 195 200 205 Leu Ala Ser Pro AlaTyr Leu Ser Leu Ser Ser Glu Asp Pro Val Leu 210 215 220 Thr Ala Leu GluLeu Ser Asn Glu Leu Ala Lys Leu Ala Asn Ile Glu 225 230 235 240 Lys GluPhe Lys Asn Asp Tyr Arg Lys Leu Ser Met Gln Cys Lys Asp 245 250 255 PheVal Val Gly Val Leu Asp Leu Cys Arg Asp Ser Glu Glu Val Glu 260 265 270Ala Ile Leu Asn Gly Asp Leu Glu Ser Ala Glu Pro Leu Glu Arg His 275 280285 Gly His Lys Ala Ser Leu Ser Arg Val Lys Leu Ala Ile Lys Tyr Glu 290295 300 Val Lys Lys Phe Val Ala His Pro Asn Cys Gln Gln Gln Leu Leu Thr305 310 315 320 Ile Trp Tyr Glu Asn Leu Ser Gly Leu Arg Glu Gln Thr IleAla Ile 325 330 335 Lys Cys Leu Val Val Leu Val Val Ala Leu Gly Leu ProPhe Leu Ala 340 345 350 Ile Gly Tyr Trp Ile Ala Pro Cys Ser Arg Leu GlyLys Ile Leu Arg 355 360 365 Ser Pro Phe Met Lys Phe Val Ala His Ala AlaSer Phe Ile Ile Phe 370 375 380 Leu Gly Leu Leu Val Phe Asn Ala Ser AspArg Phe Glu Gly Ile Thr 385 390 395 400 Thr Leu Pro Asn Ile Thr Val IleAsp Tyr Pro Lys Gln Ile Phe Arg 405 410 415 Val Lys Thr Thr Gln Phe ThrTrp Thr Glu Met Leu Ile Met Val Trp 420 425 430 Val Leu Gly Met Met TrpSer Glu Cys Lys Glu Leu Trp Leu Glu Gly 435 440 445 Pro Arg Glu Tyr IleVal Gln Leu Trp Asn Val Leu Asp Phe Gly Met 450 455 460 Leu Ser Ile PheIle Ala Ala Phe Thr Ala Arg Phe Leu Ala Phe Leu 465 470 475 480 Gln AlaThr Lys Ala Gln Gln Tyr Val Asp Ser His Val Gln Glu Ser 485 490 495 AspLeu Ser Glu Val Thr Leu Pro Pro Glu Val Gln Tyr Phe Thr Tyr 500 505 510Ala Arg Asp Lys Trp Leu Pro Ser Asp Pro Gln Ile Ile Ser Glu Gly 515 520525 Leu Tyr Ala Ile Ala Val Val Leu Ser Phe Ser Arg Ile Ala Tyr Ile 530535 540 Leu Pro Ala Asn Glu Ser Phe Gly Pro Leu Gln Ile Ser Leu Gly Arg545 550 555 560 Thr Val Lys Asp Ile Phe Lys Phe Met Val Leu Phe Ile MetVal Phe 565 570 575 Leu Ala Phe Met Ile Gly Met Phe Ile Leu Tyr Ser TyrTyr Leu Gly 580 585 590 Ala Lys Val Asn Pro Ala Phe Thr Thr Val Glu GluSer Phe Lys Thr 595 600 605 Leu Phe Trp Ser Ile Phe Gly Leu Ser Glu ValThr Ser Val Val Leu 610 615 620 Lys Tyr Asp His Lys Phe Ile Glu Asn IleGly Tyr Val Leu Tyr Gly 625 630 635 640 Ile Tyr Asn Val Thr Met Val ValVal Leu Leu Asn Met Leu Ile Ala 645 650 655 Met Ile Asn Ser Ser Tyr GlnGlu Ile Glu Asp Asp Ser Asp Val Glu 660 665 670 Trp Lys Phe Ala Arg SerLys Leu Trp Leu Ser Tyr Phe Asp Asp Gly 675 680 685 Lys Thr Leu Pro ProPro Phe Ser Leu Val Pro Ser Pro Lys Ser Phe 690 695 700 Val Tyr Phe IleMet Arg Ile Thr Asn Phe Ser Lys Cys Arg Arg Arg 705 710 715 720 Arg LeuGln Lys Asp Leu Glu Leu Gly Met Gly Asn Ser Lys Ser Arg 725 730 735 LeuAsn Leu Phe Thr Gln Ser Asn Ser Arg Val Phe Glu Ser His Ser 740 745 750Phe Asn Ser Ile Leu Asn Gln Pro Thr Arg Tyr Gln Gln Ile Met Lys 755 760765 Arg Leu Ile Lys Arg Tyr Val Leu Lys Ala Gln Val Asp Lys Glu Asn 770775 780 Asp Glu Val Asn Glu Gly Glu Leu Lys Glu Ile Lys Gln Asp Ile Ser785 790 795 800 Ser Leu Arg Tyr Glu Leu Leu Glu Asp Lys Ser Gln Ala ThrGlu Glu 805 810 815 Leu Ala Ile Leu Ile His Lys Leu Ser Glu Lys Leu AsnPro Ser Val 820 825 830 Leu Arg Cys Glu 835 104 1503 PRT Homo sapiens104 Met Glu Pro Ser Ala Leu Arg Lys Ala Gly Ser Glu Gln Glu Glu Gly 1 510 15 Phe Glu Gly Leu Pro Arg Arg Val Thr Asp Leu Gly Met Val Ser Asn 2025 30 Leu Arg Arg Ser Asn Ser Ser Leu Phe Lys Ser Trp Arg Leu Gln Cys 3540 45 Pro Phe Gly Asn Asn Asp Lys Gln Glu Ser Leu Ser Ser Trp Ile Pro 5055 60 Glu Asn Ile Lys Lys Lys Glu Cys Val Tyr Phe Val Glu Ser Ser Lys 6570 75 80 Leu Ser Asp Ala Gly Lys Val Val Cys Gln Cys Gly Tyr Thr His Glu85 90 95 Gln His Leu Glu Glu Ala Thr Lys Pro His Thr Phe Gln Gly Thr Gln100 105 110 Trp Asp Pro Lys Lys His Val Gln Glu Met Pro Thr Asp Ala PheGly 115 120 125 Asp Ile Val Phe Thr Gly Leu Ser Gln Lys Val Lys Lys TyrVal Arg 130 135 140 Val Ser Gln Asp Thr Pro Ser Ser Val Ile Tyr His LeuMet Thr Gln 145 150 155 160 His Trp Gly Leu Asp Val Pro Asn Leu Leu IleSer Val Thr Gly Gly 165 170 175 Ala Lys Asn Phe Asn Met Lys Pro Arg LeuLys Ser Ile Phe Arg Arg 180 185 190 Gly Leu Val Lys Val Ala Gln Thr ThrGly Ala Trp Ile Ile Thr Gly 195 200 205 Gly Ser His Thr Gly Val Met LysGln Val Gly Glu Ala Val Arg Asp 210 215 220 Phe Ser Leu Ser Ser Ser TyrLys Glu Gly Glu Leu Ile Thr Ile Gly 225 230 235 240 Val Ala Thr Trp GlyThr Val His Arg Arg Glu Gly Leu Ile His Pro 245 250 255 Thr Gly Ser PhePro Ala Glu Tyr Ile Leu Asp Glu Asp Gly Gln Gly 260 265 270 Asn Leu ThrCys Leu Asp Ser Asn His Ser His Phe Ile Leu Val Asp 275 280 285 Asp GlyThr His Gly Gln Tyr Gly Val Glu Ile Pro Leu Arg Thr Arg 290 295 300 LeuGlu Lys Phe Ile Ser Glu Gln Thr Lys Glu Arg Gly Gly Val Ala 305 310 315320 Ile Lys Ile Pro Ile Val Cys Val Val Leu Glu Gly Gly Pro Gly Thr 325330 335 Leu His Thr Ile Asp Asn Ala Thr Thr Asn Gly Thr Pro Cys Val Val340 345 350 Val Glu Gly Ser Gly Arg Val Ala Asp Val Ile Ala Gln Val AlaAsn 355 360 365 Leu Pro Val Ser Asp Ile Thr Ile Ser Leu Ile Gln Gln LysLeu Ser 370 375 380 Val Phe Phe Gln Glu Met Phe Glu Thr Phe Thr Glu SerArg Ile Val 385 390 395 400 Glu Trp Thr Lys Lys Ile Gln Asp Ile Val ArgArg Arg Gln Leu Leu 405 410 415 Thr Val Phe Arg Glu Gly Lys Asp Gly GlnGln Asp Val Asp Val Ala 420 425 430 Ile Leu Gln Ala Leu Leu Lys Ala SerArg Ser Gln Asp His Phe Gly 435 440 445 His Glu Asn Trp Asp His Gln LeuLys Leu Ala Val Ala Trp Asn Arg 450 455 460 Val Asp Ile Ala Arg Ser GluIle Phe Met Asp Glu Trp Gln Trp Lys 465 470 475 480 Pro Ser Asp Leu HisPro Thr Met Thr Ala Ala Leu Ile Ser Asn Lys 485 490 495 Pro Glu Phe ValLys Leu Phe Leu Glu Asn Gly Val Gln Leu Lys Glu 500 505 510 Phe Val ThrTrp Asp Thr Leu Leu Tyr Leu Tyr Glu Asn Leu Asp Pro 515 520 525 Ser CysLeu Phe His Ser Lys Leu Gln Lys Val Leu Val Glu Asp Pro 530 535 540 GluArg Pro Ala Cys Ala Pro Ala Ala Pro Arg Leu Gln Met His His 545 550 555560 Val Ala Gln Val Leu Arg Glu Leu Leu Gly Asp Phe Thr Gln Pro Leu 565570 575 Tyr Pro Arg Pro Arg His Asn Asp Arg Leu Arg Leu Leu Leu Pro Val580 585 590 Pro His Val Lys Leu Asn Val Gln Gly Val Ser Leu Arg Ser LeuTyr 595 600 605 Lys Arg Ser Ser Gly His Val Thr Phe Thr Met Asp Pro IleArg Asp 610 615 620 Leu Leu Ile Trp Ala Ile Val Gln Asn Arg Arg Glu LeuAla Gly Ile 625 630 635 640 Ile Trp Ala Gln Ser Gln Asp Cys Ile Ala AlaAla Leu Ala Cys Ser 645 650 655 Lys Ile Leu Lys Glu Leu Ser Lys Glu GluGlu Asp Thr Asp Ser Ser 660 665 670 Glu Glu Met Leu Ala Leu Ala Glu GluTyr Glu His Arg Ala Ile Gly 675 680 685 Val Phe Thr Glu Cys Tyr Arg LysAsp Glu Glu Arg Ala Gln Lys Leu 690 695 700 Leu Thr Arg Val Ser Glu AlaTrp Gly Lys Thr Thr Cys Leu Gln Leu 705 710 715 720 Ala Leu Glu Ala LysAsp Met Lys Phe Val Ser His Gly Gly Ile Gln 725 730 735 Ala Phe Leu ThrLys Val Trp Trp Gly Gln Leu Ser Val Asp Asn Gly 740 745 750 Leu Trp ArgVal Thr Leu Cys Met Leu Ala Phe Pro Leu Leu Leu Thr 755 760 765 Gly LeuIle Ser Phe Arg Glu Lys Arg Leu Gln Asp Val Gly Thr Pro 770 775 780 AlaAla Arg Ala Arg Ala Phe Phe Thr Ala Pro Val Val Val Phe His 785 790 795800 Leu Asn Ile Leu Ser Tyr Phe Ala Phe Leu Cys Leu Phe Ala Tyr Val 805810 815 Leu Met Val Asp Phe Gln Pro Val Pro Ser Trp Cys Glu Cys Ala Ile820 825 830 Tyr Leu Trp Leu Phe Ser Leu Val Cys Glu Glu Met Arg Gln LeuPhe 835 840 845 Tyr Asp Pro Asp Glu Cys Gly Leu Met Lys Lys Ala Ala LeuTyr Phe 850 855 860 Ser Asp Phe Trp Asn Lys Leu Asp Val Gly Ala Ile LeuLeu Phe Val 865 870 875 880 Ala Gly Leu Thr Cys Arg Leu Ile Pro Ala ThrLeu Tyr Pro Gly Arg 885 890 895 Val Ile Leu Ser Leu Asp Phe Ile Leu PheCys Leu Arg Leu Met His 900 905 910 Ile Phe Thr Ile Ser Lys Thr Leu GlyPro Lys Ile Ile Ile Val Lys 915 920 925 Arg Met Met Lys Asp Val Phe PhePhe Leu Phe Leu Leu Ala Val Trp 930 935 940 Val Val Ser Phe Gly Val AlaLys Gln Ala Ile Leu Ile His Asn Glu 945 950 955 960 Arg Arg Val Asp TrpLeu Phe Arg Gly Ala Val Tyr His Ser Tyr Leu 965 970 975 Thr Ile Phe GlyGln Ile Pro Gly Tyr Ile Asp Gly Val Asn Phe Asn 980 985 990 Pro Glu HisCys Ser Pro Asn Gly Thr Asp Pro Tyr Lys Pro Lys Cys 995 1000 1005 ProGlu Ser Asp Ala Thr Gln Gln Arg Pro Ala Phe Pro Glu Trp 1010 1015 1020Leu Thr Val Leu Leu Leu Cys Leu Tyr Leu Leu Phe Thr Asn Ile 1025 10301035 Leu Leu Leu Asn Leu Leu Ile Ala Met Phe Asn Tyr Thr Phe Gln 10401045 1050 Gln Val Gln Glu His Thr Asp Gln Ile Trp Lys Phe Gln Arg His1055 1060 1065 Asp Leu Ile Glu Glu Tyr His Gly Arg Pro Ala Ala Pro ProPro 1070 1075 1080 Phe Ile Leu Leu Ser His Leu Gln Leu Phe Ile Lys ArgVal Val 1085 1090 1095 Leu Lys Thr Pro Ala Lys Arg His Lys Gln Leu LysAsn Lys Leu 1100 1105 1110 Glu Lys Asn Glu Glu Ala Ala Leu Leu Ser TrpGlu Ile Tyr Leu 1115 1120 1125 Lys Glu Asn Tyr Leu Gln Asn Arg Gln PheGln Gln Lys Gln Arg 1130 1135 1140 Pro Glu Gln Lys Ile Glu Asp Ile SerAsn Lys Val Asp Ala Met 1145 1150 1155 Val Asp Leu Leu Asp Leu Asp ProLeu Lys Arg Ser Gly Ser Met 1160 1165 1170 Glu Gln Arg Leu Ala Ser LeuGlu Glu Gln Val Ala Gln Thr Ala 1175 1180 1185 Arg Ala Leu His Trp IleVal Arg Thr Leu Arg Ala Ser Gly Phe 1190 1195 1200 Ser Ser Glu Ala AspVal Pro Thr Leu Ala Ser Gln Lys Ala Ala 1205 1210 1215 Glu Glu Pro AspAla Glu Pro Gly Gly Arg Lys Lys Thr Glu Glu 1220 1225 1230 Pro Gly AspSer Tyr His Val Asn Ala Arg His Leu Leu Tyr Pro 1235 1240 1245 Asn CysPro Val Thr Arg Phe Pro Val Pro Asn Glu Lys Val Pro 1250 1255 1260 TrpGlu Thr Glu Phe Leu Ile Tyr Asp Pro Pro Phe Tyr Thr Ala 1265 1270 1275Glu Arg Lys Asp Ala Ala Ala Met Asp Pro Met Gly Asp Thr Leu 1280 12851290 Glu Pro Leu Ser Thr Ile Gln Tyr Asn Val Val Asp Gly Leu Arg 12951300 1305 Asp Arg Arg Ser Phe His Gly Pro Tyr Thr Val Gln Ala Gly Leu1310 1315 1320 Pro Leu Asn Pro Met Gly Arg Thr Gly Leu Arg Gly Arg GlySer 1325 1330 1335 Leu Ser Cys Phe Gly Pro Asn His Thr Leu Tyr Pro MetVal Thr 1340 1345 1350 Arg Trp Arg Arg Asn Glu Asp Gly Ala Ile Cys ArgLys Ser Ile 1355 1360 1365 Lys Lys Met Leu Glu Val Leu Val Val Lys LeuPro Leu Ser Glu 1370 1375 1380 His Trp Ala Leu Pro Gly Gly Ser Arg GluPro Gly Glu Met Leu 1385 1390 1395 Pro Arg Lys Leu Lys Arg Ile Leu ArgGln Glu His Trp Pro Ser 1400 1405 1410 Phe Glu Asn Leu Leu Lys Cys GlyMet Glu Val Tyr Lys Gly Tyr 1415 1420 1425 Met Asp Asp Pro Arg Asn ThrAsp Asn Ala Trp Ile Glu Thr Val 1430 1435 1440 Ala Val Ser Val His PheGln Asp Gln Asn Asp Val Glu Leu Asn 1445 1450 1455 Arg Leu Asn Ser AsnLeu His Ala Cys Asp Ser Gly Ala Ser Ile 1460 1465 1470 Arg Trp Gln ValVal Asp Arg Arg Ile Pro Leu Tyr Ala Asn His 1475 1480 1485 Lys Thr LeuLeu Gln Lys Ala Ala Ala Glu Phe Gly Ala His Tyr 1490 1495 1500 105 966PRT Mus musculus 105 Met Ala Gln Leu Tyr Tyr Lys Lys Val Asn Tyr Ser ProTyr Arg Asp 1 5 10 15 Arg Ile Pro Leu Gln Ile Val Arg Ala Glu Thr GluLeu Ser Ala Glu 20 25 30 Glu Lys Ala Phe Leu Ser Ala Val Glu Lys Gly AspTyr Ala Thr Val 35 40 45 Lys Gln Ala Leu Gln Glu Ala Glu Ile Tyr Tyr AsnVal Asn Ile Asn 50 55 60 Cys Met Asp Pro Leu Gly Arg Ser Ala Leu Leu IleAla Ile Glu Asn 65 70 75 80 Glu Asn Leu Glu Ile Met Glu Leu Leu Leu AsnHis Ser Val Tyr Val 85 90 95 Gly Asp Ala Leu Leu Tyr Ala Ile Arg Lys GluVal Val Gly Ala Val 100 105 110 Glu Leu Leu Leu Ser Tyr Arg Lys Pro SerGly Glu Lys Gln Val Pro 115 120 125 Thr Leu Met Met Asp Thr Gln Phe SerGlu Phe Thr Pro Asp Ile Thr 130 135 140 Pro Ile Met Leu Ala Ala His ThrAsn Asn Tyr Glu Ile Ile Lys Leu 145 150 155 160 Leu Val Gln Lys Arg ValThr Ile Pro Arg Pro His Gln Ile Arg Cys 165 170 175 Asn Cys Val Glu CysVal Ser Ser Ser Glu Val Asp Ser Leu Arg His 180 185 190 Ser Arg Ser ArgLeu Asn Ile Tyr Lys Ala Leu Ala Ser Pro Ser Leu 195 200 205 Ile Ala LeuSer Ser Glu Asp Pro Ile Leu Thr Ala Phe Arg Leu Gly 210 215 220 Trp GluLeu Lys Glu Leu Ser Lys Val Glu Asn Glu Phe Lys Ala Glu 225 230 235 240Tyr Glu Glu Leu Ser Gln Gln Cys Lys Leu Phe Ala Lys Asp Leu Leu 245 250255 Asp Gln Ala Arg Ser Ser Arg Glu Leu Glu Ile Ile Leu Asn His Arg 260265 270 Asp Asp His Ser Glu Glu Leu Asp Pro Gln Lys Tyr His Asp Leu Ala275 280 285 Lys Leu Lys Val Ala Ile Lys Tyr His Gln Lys Glu Phe Val AlaGln 290 295 300 Pro Asn Cys Gln Gln Leu Leu Ala Thr Leu Trp Tyr Asp GlyPhe Pro 305 310 315 320 Gly Trp Arg Arg Lys His Trp Val Val Lys Leu LeuThr Cys Met Thr 325 330 335 Ile Gly Phe Leu Phe Pro Met Leu Ser Ile AlaTyr Leu Ile Ser Pro 340 345 350 Arg Ser Asn Leu Gly Leu Phe Ile Lys LysPro Phe Ile Lys Phe Ile 355 360 365 Cys His Thr Ala Ser Tyr Leu Thr PheLeu Phe Met Leu Leu Leu Ala 370 375 380 Ser Gln His Ile Val Arg Thr AspLeu His Val Gln Gly Pro Pro Pro 385 390 395 400 Thr Val Val Glu Trp MetIle Leu Pro Trp Val Leu Gly Phe Ile Trp 405 410 415 Gly Glu Ile Lys GluMet Trp Asp Gly Gly Phe Thr Glu Tyr Ile His 420 425 430 Asp Trp Trp AsnLeu Met Asp Phe Ala Met Asn Ser Leu Tyr Leu Ala 435 440 445 Thr Ile SerLeu Lys Ile Val Ala Tyr Val Lys Tyr Asn Gly Ser Arg 450 455 460 Pro ArgGlu Glu Trp Glu Met Trp His Pro Thr Leu Ile Ala Glu Ala 465 470 475 480Leu Phe Ala Ile Ser Asn Ile Leu Ser Ser Leu Arg Leu Ile Ser Leu 485 490495 Phe Thr Ala Asn Ser His Leu Gly Pro Leu Gln Ile Ser Leu Gly Arg 500505 510 Met Leu Leu Asp Ile Leu Lys Phe Leu Phe Ile Tyr Cys Leu Val Leu515 520 525 Leu Ala Phe Ala Asn Gly Leu Asn Gln Leu Tyr Phe Tyr Tyr GluThr 530 535 540 Arg Ala Ile Asp Glu Pro Asn Asn Cys Lys Gly Ile Arg CysGlu Lys 545 550 555 560 Gln Asn Asn Ala Phe Ser Thr Leu Phe Glu Thr LeuGln Ser Leu Phe 565 570 575 Trp Ser Val Phe Gly Leu Leu Asn Leu Tyr ValThr Asn Val Lys Ala 580 585 590 Arg His Glu Phe Thr Glu Phe Val Gly AlaThr Met Phe Gly Thr Tyr 595 600 605 Asn Val Ile Ser Leu Val Val Leu LeuAsn Met Leu Ile Ala Met Met 610 615 620 Asn Asn Ser Tyr Gln Leu Ile AlaAsp His Ala Asp Ile Glu Trp Lys 625 630 635 640 Phe Ala Arg Thr Lys LeuTrp Met Ser Tyr Phe Asp Glu Gly Gly Thr 645 650 655 Leu Pro Pro Pro PheAsn Ile Ile Pro Ser Pro Lys Ser Phe Leu Tyr 660 665 670 Leu Gly Asn TrpPhe Asn Asn Thr Phe Cys Pro Lys Arg Asp Pro Asp 675 680 685 Gly Arg ArgArg Arg His Asn Leu Arg Ser Phe Thr Glu Arg His Ala 690 695 700 Asp SerLeu Ile Gln Asn Gln His Tyr Gln Glu Val Ile Arg Asn Leu 705 710 715 720Val Lys Arg Tyr Val Ala Ala Met Ile Arg Asn Ser Lys Thr Asn Glu 725 730735 Gly Leu Thr Glu Glu Asn Phe Lys Glu Leu Lys Gln Asp Ile Ser Ser 740745 750 Phe Arg Tyr Glu Val Leu Asp Leu Leu Gly Asn Arg Lys His Pro Arg755 760 765 Arg Ser Leu Ser Thr Ser Ser Ala Asp Phe Ser Gln Arg Asp AspThr 770 775 780 Asn Asp Gly Ser Gly Gly Ala Arg Ala Lys Ser Lys Ser ValSer Phe 785 790 795 800 Asn Val Gly Cys Lys Lys Lys Ala Cys His Gly AlaPro Leu Ile Arg 805 810 815 Thr Val Pro Arg Ala Ser Gly Ala Gln Gly LysPro Lys Ser Glu Ser 820 825 830 Ser Ser Lys Arg Ser Phe Met Gly Pro SerPhe Lys Lys Leu Gly Leu 835 840 845 Phe Phe Ser Lys Phe Asn Gly Gln ThrSer Glu Pro Thr Ser Glu Pro 850 855 860 Met Tyr Thr Ile Ser Asp Gly IleAla Gln Gln His Cys Met Trp Gln 865 870 875 880 Asp Ile Arg Tyr Ser GlnMet Glu Lys Gly Lys Ala Glu Ala Cys Ser 885 890 895 Gln Ser Gln Met AsnLeu Gly Glu Val Glu Leu Gly Glu Ile Arg Gly 900 905 910 Ala Ala Ala ArgSer Ser Glu Cys Pro Leu Ala Cys Ser Ser Ser Leu 915 920 925 His Cys AlaSer Gly Ile Cys Ser Ser Asn Ser Lys Leu Leu Asp Ser 930 935 940 Ser GluAsp Val Phe Glu Thr Trp Gly Glu Ala Cys Asp Leu Leu Met 945 950 955 960His Lys Trp Gly Asp Gly 965 106 974 PRT Mus musculus 106 Met Ala Gln PheTyr Tyr Lys Arg Asn Val Asn Ala Pro Tyr Arg Asp 1 5 10 15 Arg Ile ProLeu Arg Ile Val Arg Ala Glu Ser Glu Leu Ser Pro Ser 20 25 30 Glu Lys AlaTyr Leu Asn Ala Val Glu Lys Gly Asp Tyr Ala Ser Val 35 40 45 Lys Lys SerLeu Glu Glu Ala Glu Ile Tyr Phe Lys Ile Asn Ile Asn 50 55 60 Cys Ile AspPro Leu Gly Arg Thr Ala Leu Leu Ile Ala Ile Glu Asn 65 70 75 80 Glu AsnLeu Glu Leu Ile Glu Leu Leu Leu Ser Phe Asn Val Tyr Val 85 90 95 Gly AspAla Leu Leu His Ala Ile Arg Lys Glu Val Val Gly Ala Val 100 105 110 GluLeu Leu Leu Asn His Lys Lys Pro Ser Gly Glu Lys Gln Val Pro 115 120 125Pro Ile Leu Leu Asp Lys Gln Phe Ser Glu Phe Thr Pro Asp Ile Thr 130 135140 Pro Ile Ile Leu Ala Ala His Thr Asn Asn Tyr Glu Ile Ile Lys Leu 145150 155 160 Leu Val Gln Lys Gly Val Ser Val Pro Arg Pro His Glu Val ArgCys 165 170 175 Asn Cys Val Glu Cys Val Ser Ser Ser Asp Val Asp Ser LeuArg His 180 185 190 Ser Arg Ser Arg Leu Asn Ile Tyr Lys Ala Leu Ala SerPro Ser Leu 195 200 205 Ile Ala Leu Ser Ser Glu Asp Pro Phe Leu Thr AlaPhe Gln Leu Ser 210 215 220 Trp Glu Leu Gln Glu Leu Ser Lys Val Glu AsnGlu Phe Lys Ser Glu 225 230 235 240 Tyr Glu Glu Leu Ser Arg Gln Cys LysGln Phe Ala Lys Asp Leu Leu 245 250 255 Asp Gln Thr Arg Ser Ser Arg GluLeu Glu Ile Ile Leu Asn Tyr Arg 260 265 270 Asp Asp Asn Ser Leu Ile GluGlu Gln Ser Gly Asn Asp Leu Ala Arg 275 280 285 Leu Lys Leu Ala Ile LysTyr Arg Gln Lys Glu Phe Val Ala Gln Pro 290 295 300 Asn Cys Gln Gln LeuLeu Ala Ser Arg Trp Tyr Asp Glu Phe Pro Gly 305 310 315 320 Trp Arg ArgArg His Trp Ala Val Lys Met Val Thr Cys Phe Ile Ile 325 330 335 Gly LeuLeu Phe Pro Val Phe Ser Val Cys Tyr Leu Ile Ala Pro Lys 340 345 350 SerPro Leu Gly Leu Phe Ile Arg Lys Pro Phe Ile Lys Phe Ile Cys 355 360 365His Thr Ala Ser Tyr Leu Thr Phe Leu Phe Leu Leu Leu Leu Ala Ser 370 375380 Gln His Ile Asp Arg Ser Asp Leu Asn Arg Gln Gly Pro Pro Pro Thr 385390 395 400 Ile Val Glu Trp Met Ile Leu Pro Trp Val Leu Gly Phe Ile TrpGly 405 410 415 Glu Ile Lys Gln Met Trp Asp Gly Gly Leu Gln Asp Tyr IleHis Asp 420 425 430 Trp Trp Asn Leu Met Asp Phe Val Met Asn Ser Leu TyrLeu Ala Thr 435 440 445 Ile Ser Leu Lys Ile Val Ala Phe Val Lys Tyr SerAla Leu Asn Pro 450 455 460 Arg Glu Ser Trp Asp Met Trp His Pro Thr LeuVal Ala Glu Ala Leu 465 470 475 480 Phe Ala Ile Ala Asn Ile Phe Ser SerLeu Arg Leu Ile Ser Leu Phe 485 490 495 Thr Ala Asn Ser His Leu Gly ProLeu Gln Ile Ser Leu Gly Arg Met 500 505 510 Leu Leu Asp Ile Leu Lys PheLeu Phe Ile Tyr Cys Leu Val Leu Leu 515 520 525 Ala Phe Ala Asn Gly LeuAsn Gln Leu Tyr Phe Tyr Tyr Glu Glu Thr 530 535 540 Lys Gly Leu Ser CysLys Gly Ile Arg Cys Glu Lys Gln Asn Asn Ala 545 550 555 560 Phe Ser ThrLeu Phe Glu Thr Leu Gln Ser Leu Phe Trp Ser Ile Phe 565 570 575 Gly LeuIle Asn Leu Tyr Val Thr Asn Val Lys Ala Gln His Glu Phe 580 585 590 ThrGlu Phe Val Gly Ala Thr Met Phe Gly Thr Tyr Asn Val Ile Ser 595 600 605Leu Val Val Leu Leu Asn Met Leu Ile Ala Met Met Asn Asn Ser Tyr 610 615620 Gln Leu Ile Ala Asp His Ala Asp Ile Glu Trp Lys Phe Ala Arg Thr 625630 635 640 Lys Leu Trp Met Ser Tyr Phe Glu Glu Gly Gly Thr Leu Pro ThrPro 645 650 655 Phe Asn Val Ile Pro Ser Pro Lys Ser Leu Trp Tyr Leu ValLys Trp 660 665 670 Ile Trp Thr His Leu Cys Lys Lys Lys Met Arg Arg LysPro Glu Ser 675 680 685 Phe Gly Thr Ile Gly Arg Arg Ala Ala Asp Asn LeuArg Arg His His 690 695 700 Gln Tyr Gln Glu Val Met Arg Asn Leu Val LysArg Tyr Val Ala Ala 705 710 715 720 Met Ile Arg Glu Ala Lys Thr Glu GluGly Leu Thr Glu Glu Asn Val 725 730 735 Lys Glu Leu Lys Gln Asp Ile SerSer Phe Arg Phe Glu Val Leu Gly 740 745 750 Leu Leu Arg Gly Ser Lys LeuSer Thr Ile Gln Ser Ala Asn Ala Ala 755 760 765 Ser Ser Ala Asp Ser AspGlu Lys Ser Gln Ser Glu Gly Asn Gly Lys 770 775 780 Asp Lys Arg Lys AsnLeu Ser Leu Phe Asp Leu Thr Thr Leu Ile His 785 790 795 800 Pro Arg SerAla Ala Ile Ala Ser Glu Arg His Asn Leu Ser Asn Gly 805 810 815 Ser AlaLeu Val Val Gln Glu Pro Pro Arg Glu Lys Gln Arg Lys Val 820 825 830 AsnPhe Val Ala Asp Ile Lys Asn Phe Gly Leu Phe His Arg Arg Ser 835 840 845Lys Gln Asn Ala Ala Glu Gln Asn Ala Asn Gln Ile Phe Ser Val Ser 850 855860 Glu Glu Ile Thr Arg Gln Gln Ala Ala Gly Ala Leu Glu Arg Asn Ile 865870 875 880 Glu Leu Glu Ser Lys Gly Leu Ala Ser Arg Gly Asp Arg Ser IlePro 885 890 895 Gly Leu Asn Glu Gln Cys Val Leu Val Asp His Arg Glu ArgAsn Thr 900 905 910 Asp Thr Leu Gly Leu Gln Val Gly Lys Arg Val Cys SerThr Phe Lys 915 920 925 Ser Glu Lys Val Val Val Glu Asp Thr Val Pro IleIle Pro Lys Glu 930 935 940 Lys His Ala His Glu Glu Asp Ser Ser Ile AspTyr Asp Leu Ser Pro 945 950 955 960 Thr Asp Thr Ala Ala His Glu Asp TyrVal Thr Thr Arg Leu 965 970 107 1072 PRT Mus musculus 107 Met Gly ThrLys Thr His Pro Val Val Pro Trp Ser Thr Lys Glu Ile 1 5 10 15 Ser GluLeu Lys Gly Met Leu Lys Gln Leu Gln Pro Gly Pro Leu Gly 20 25 30 Arg AlaAla Arg Met Val Leu Ser Ala Ala Arg Lys Ala Pro Pro Ala 35 40 45 Ser ValVal Ser Pro Asn Asn Ser His Gly Glu Pro Gly Pro Ser Arg 50 55 60 Ala GluSer Ala Glu Pro Arg Ala Glu Glu Pro Asn Arg Lys Thr Ala 65 70 75 80 ValGly Arg Arg Lys Arg Arg Lys Val Gln Glu Pro Arg Arg Ser Leu 85 90 95 SerAsn Ser Ser Ser Gln Pro Asn Arg Arg Thr Gly Arg Thr Arg Gln 100 105 110Arg Gln His Arg Pro Gln Thr Lys Ser Asp Asp Gly Gly Val Gln Ala 115 120125 Ala Gly Gln Cys Pro Ile Cys Ala Gly Phe Phe Ser Ile Glu Thr Leu 130135 140 Pro Gln His Ala Ala Thr Cys Gly Glu Ser Pro Pro Pro Gln Pro Ala145 150 155 160 Ser Pro Ala Ser Leu Ser Ser Ser Glu Ser Val Leu Arg ArgHis His 165 170 175 Val Ala Leu Thr Pro Val Pro Leu Val Pro Lys Pro GlnPro Asn Trp 180 185 190 Thr Glu Ile Val Asn Lys Lys Leu Lys Phe Pro ProThr Leu Leu Arg 195 200 205 Ala Ile Gln Glu Gly Gln Leu Gly Leu Val GlnGln Leu Leu Glu Ser 210 215 220 Ser Ser Asp Ala Ser Gly Ala Gly Pro GlyGly Pro Leu Arg Asn Val 225 230 235 240 Glu Glu Ser Glu Asp Arg Ser TrpArg Glu Ala Leu Asn Leu Ala Ile 245 250 255 Arg Leu Gly His Glu Val IleThr Asp Val Leu Leu Ala Asn Val Lys 260 265 270 Phe Asp Phe Arg Gln IleHis Glu Ala Leu Leu Val Ala Val Asp Thr 275 280 285 Asn Gln Pro Ala ValVal Arg Arg Leu Leu Ala Arg Leu Glu Arg Glu 290 295 300 Lys Gly Arg LysVal Asp Thr Lys Ser Phe Ser Leu Ala Phe Phe Asp 305 310 315 320 Ser SerIle Asp Gly Ser Arg Phe Ala Pro Gly Val Thr Pro Leu Thr 325 330 335 LeuAla Cys Gln Lys Asp Leu Tyr Glu Ile Ala Gln Leu Leu Met Asp 340 345 350Gln Gly His Thr Ile Ala Arg Pro His Pro Val Ser Cys Ala Cys Leu 355 360365 Glu Cys Ser Asn Ala Arg Arg Tyr Asp Leu Leu Lys Phe Ser Leu Ser 370375 380 Arg Ile Asn Thr Tyr Arg Gly Ile Ala Ser Arg Ala His Leu Ser Leu385 390 395 400 Ala Ser Glu Asp Ala Met Leu Ala Ala Phe Gln Leu Ser ArgGlu Leu 405 410 415 Arg Arg Leu Ala Arg Lys Glu Pro Glu Phe Lys Pro GlnTyr Ile Ala 420 425 430 Leu Glu Ser Leu Cys Gln Asp Tyr Gly Phe Glu LeuLeu Gly Met Cys 435 440 445 Arg Asn Gln Ser Glu Val Thr Ala Val Leu AsnAsp Leu Gly Glu Asp 450 455 460 Ser Glu Thr Glu Pro Glu Ala Glu Gly LeuGly Gln Ala Phe Glu Glu 465 470 475 480 Gly Ile Pro Asn Leu Ala Arg LeuArg Leu Ala Val Asn Tyr Asn Gln 485 490 495 Lys Gln Phe Val Ala His ProIle Cys Gln Gln Val Leu Ser Ser Ile 500 505 510 Trp Cys Gly Asn Leu AlaGly Trp Arg Gly Ser Thr Thr Ile Trp Arg 515 520 525 Leu Phe Val Ala SerLeu Ile Phe Leu Thr Met Pro Phe Leu Cys Ile 530 535 540 Gly Tyr Trp LeuAla Pro Lys Ser Gln Leu Gly Arg Leu Leu Lys Ile 545 550 555 560 Pro ValLeu Lys Phe Leu Leu His Ser Ala Ser Tyr Leu Trp Phe Leu 565 570 575 IlePhe Leu Leu Gly Glu Ser Leu Val Met Glu Thr Gln Leu Ser Thr 580 585 590Phe Lys Gly Arg Ser Gln Ser Val Trp Glu Thr Ser Leu His Met Ile 595 600605 Trp Val Thr Gly Phe Leu Trp Phe Glu Cys Lys Glu Val Trp Ile Glu 610615 620 Gly Leu Arg Ser Tyr Leu Leu Asp Trp Trp Asn Phe Leu Asp Val Val625 630 635 640 Ile Leu Ser Leu Tyr Leu Ala Ser Phe Ala Leu Arg Leu LeuLeu Ala 645 650 655 Gly Leu Ala Tyr Met His Cys Arg Asp Ala Ser Asp SerThr Thr Cys 660 665 670 Arg Cys Phe Thr Thr Ala Glu Arg Ser Glu Trp ArgThr Glu Asp Pro 675 680 685 Gln Phe Leu Ala Glu Val Leu Phe Thr Val ThrSer Met Leu Ser Phe 690 695 700 Thr Arg Leu Ala Tyr Ile Leu Pro Ala HisGlu Ser Leu Gly Thr Leu 705 710 715 720 Gln Ile Ser Ile Gly Lys Met IleAsp Asp Met Ile Arg Phe Met Phe 725 730 735 Ile Leu Met Ile Ile Leu ThrAla Phe Leu Cys Gly Leu Asn Asn Ile 740 745 750 Tyr Val Pro Tyr Gln GluSer Glu Lys Leu Gly Asn Phe Asn Glu Thr 755 760 765 Phe Gln Phe Leu PheTrp Thr Met Phe Gly Met Glu Glu His Thr Val 770 775 780 Val Asp Met ProGln Phe Leu Val Pro Glu Phe Val Gly Arg Ala Met 785 790 795 800 Tyr GlyIle Phe Thr Ile Val Met Val Ile Val Leu Leu Asn Met Leu 805 810 815 IleAla Met Ile Thr Asn Ser Phe Gln Lys Ile Glu Asp Asp Ala Asp 820 825 830Val Glu Trp Lys Phe Ala Arg Ser Lys Leu Tyr Leu Ser Tyr Phe Arg 835 840845 Glu Gly Leu Thr Leu Pro Val Pro Phe Asn Ile Leu Pro Ser Pro Lys 850855 860 Ala Ala Phe Tyr Leu Val Arg Arg Ile Phe Arg Phe Leu Cys Cys Gly865 870 875 880 Ser Ser Cys Cys Lys Ala Lys Lys Ser Asp Tyr Pro Pro IleGly Thr 885 890 895 Phe Thr Asn Pro Gly Ala Arg Ala Gly Ser Ala Gly GluGly Glu Arg 900 905 910 Val Ser Tyr Arg Leu Arg Val Ile Lys Ala Leu ValGln Arg Tyr Ile 915 920 925 Glu Thr Ala Arg Arg Glu Phe Glu Glu Thr ArgArg Lys Asp Leu Gly 930 935 940 Asn Arg Leu Thr Glu Leu Thr Lys Thr ValSer Arg Leu Gln Ser Glu 945 950 955 960 Val Ala Ser Val Gln Lys Asn LeuAla Ala Gly Gly Ala Pro Arg Pro 965 970 975 Pro Asp Gly Ala Ser Ile LeuSer Arg Tyr Ile Thr Arg Val Arg Asn 980 985 990 Ser Phe Gln Asn Leu GlyPro Pro Thr Ser Asp Thr Pro Ala Glu Leu 995 1000 1005 Thr Met Pro GlyIle Val Glu Thr Glu Val Ser Leu Gly Asp Gly 1010 1015 1020 Leu Asp GlyThr Gly Glu Ala Gly Ala Pro Ala Pro Gly Glu Pro 1025 1030 1035 Gly SerSer Ser Ser Ala His Val Leu Val His Arg Glu Gln Glu 1040 1045 1050 AlaGlu Gly Ser Gly Asp Leu Leu Leu Glu Gly Asp Leu Glu Thr 1055 1060 1065Lys Gly Glu Ser 1070 108 809 PRT Mus musculus 108 Met Gly Ala Pro ProPro Ser Pro Gly Leu Pro Pro Ser Trp Ala Ala 1 5 10 15 Met Met Ala AlaLeu Tyr Pro Ser Thr Asp Leu Ser Gly Val Ser Ser 20 25 30 Ser Ser Leu ProSer Ser Pro Ser Ser Ser Ser Pro Asn Glu Val Met 35 40 45 Ala Leu Lys AspVal Arg Glu Val Lys Glu Glu Asn Thr Leu Asn Glu 50 55 60 Lys Leu Phe LeuLeu Ala Cys Asp Lys Gly Asp Tyr Tyr Met Val Lys 65 70 75 80 Lys Ile LeuGlu Glu Asn Ser Ser Gly Asp Leu Asn Ile Asn Cys Val 85 90 95 Asp Val LeuGly Arg Asn Ala Val Thr Ile Thr Ile Glu Asn Glu Ser 100 105 110 Leu AspIle Leu Gln Leu Leu Leu Asp Tyr Gly Cys Gln Ser Ala Asp 115 120 125 AlaLeu Leu Val Ala Ile Asp Ser Glu Val Val Gly Ala Val Asp Ile 130 135 140Leu Leu Asn His Arg Pro Lys Arg Ser Ser Arg Pro Thr Ile Val Lys 145 150155 160 Leu Met Glu Arg Ile Gln Asn Pro Glu Tyr Ser Thr Thr Met Asp Val165 170 175 Ala Pro Val Ile Leu Ala Ala His Arg Asn Asn Tyr Glu Ile LeuThr 180 185 190 Met Leu Leu Lys Gln Asp Val Ser Leu Pro Lys Pro His AlaVal Gly 195 200 205 Cys Glu Cys Thr Leu Cys Ser Ala Lys Asn Lys Lys AspSer Leu Arg 210 215 220 His Ser Arg Phe Arg Leu Asp Ile Tyr Arg Cys LeuAla Ser Pro Ala 225 230 235 240 Leu Ile Met Leu Thr Glu Glu Asp Pro IleLeu Arg Ala Phe Glu Leu 245 250 255 Ser Ala Asp Leu Lys Glu Leu Ser LeuVal Glu Val Glu Phe Arg Asn 260 265 270 Asp Tyr Glu Glu Leu Ala Arg GlnCys Lys Met Phe Ala Lys Asp Leu 275 280 285 Leu Ala Gln Ala Arg Asn SerArg Glu Leu Glu Val Ile Leu Asn His 290 295 300 Thr Ser Ser Asp Glu ProLeu Asp Lys Arg Gly Leu Leu Glu Glu Arg 305 310 315 320 Met Asn Leu SerArg Leu Lys Leu Ala Ile Lys Tyr Asn Gln Lys Glu 325 330 335 Phe Val SerGln Ser Asn Cys Gln Gln Phe Leu Asn Thr Val Trp Phe 340 345 350 Gly GlnMet Ser Gly Tyr Arg Arg Lys Pro Thr Cys Lys Lys Ile Met 355 360 365 ThrVal Leu Thr Val Gly Ile Phe Trp Pro Val Leu Ser Leu Cys Tyr 370 375 380Leu Ile Ala Pro Lys Ser Gln Phe Gly Arg Ile Ile His Thr Pro Phe 385 390395 400 Met Lys Phe Ile Ile His Gly Ala Ser Tyr Phe Thr Phe Leu Leu Leu405 410 415 Leu Asn Leu Tyr Ser Leu Val Tyr Asn Glu Asp Lys Lys Asn ThrMet 420 425 430 Gly Pro Ala Leu Glu Arg Ile Asp Tyr Leu Leu Ile Leu TrpIle Ile 435 440 445 Gly Met Ile Trp Ser Asp Ile Lys Arg Leu Trp Tyr GluGly Leu Glu 450 455 460 Asp Phe Leu Glu Glu Ser Arg Asn Gln Leu Ser PheVal Met Asn Ser 465 470 475 480 Leu Tyr Leu Ala Thr Phe Ala Leu Lys ValVal Ala His Asn Lys Phe 485 490 495 His Asp Phe Ala Asp Arg Lys Asp TrpAsp Ala Phe His Pro Thr Leu 500 505 510 Val Ala Glu Gly Leu Phe Ala PheAla Asn Val Leu Ser Tyr Leu Arg 515 520 525 Leu Phe Phe Met Tyr Thr ThrSer Ser Ile Leu Gly Pro Leu Gln Ile 530 535 540 Ser Met Gly Gln Met LeuGln Asp Phe Gly Lys Phe Leu Gly Met Phe 545 550 555 560 Leu Leu Val LeuPhe Ser Phe Thr Ile Gly Leu Thr Gln Leu Tyr Asp 565 570 575 Lys Gly TyrThr Ser Lys Glu Gln Lys Asp Cys Val Gly Ile Phe Cys 580 585 590 Glu GlnGln Ser Asn Asp Thr Phe His Ser Phe Ile Gly Thr Cys Phe 595 600 605 AlaLeu Phe Trp Tyr Ile Phe Ser Leu Ala His Val Ala Ile Phe Val 610 615 620Thr Arg Phe Ser Tyr Gly Glu Glu Leu Gln Ser Phe Val Gly Ala Val 625 630635 640 Ile Val Gly Thr Tyr Asn Val Val Val Val Ile Val Leu Thr Lys Leu645 650 655 Leu Val Ala Met Leu His Lys Ser Phe Gln Leu Ile Ala Asn HisGlu 660 665 670 Asp Lys Glu Trp Lys Phe Ala Arg Ala Lys Leu Trp Leu SerTyr Phe 675 680 685 Asp Asp Lys Cys Thr Leu Pro Pro Pro Phe Asn Ile IlePro Ser Pro 690 695 700 Lys Thr Ile Cys Tyr Met Ile Ser Ser Leu Ser LysTrp Ile Cys Ser 705 710 715 720 His Thr Ser Lys Gly Lys Val Lys Arg GlnAsn Ser Leu Lys Glu Trp 725 730 735 Arg Asn Leu Lys Gln Lys Arg Asp GluAsn Tyr Gln Lys Val Met Cys 740 745 750 Cys Leu Val His Arg Tyr Leu ThrSer Met Arg Gln Lys Met Gln Ser 755 760 765 Thr Asp Gln Ala Thr Val GluAsn Leu Asn Glu Leu Arg Gln Asp Leu 770 775 780 Ser Lys Phe Arg Asn GluIle Arg Asp Leu Leu Gly Phe Arg Thr Ser 785 790 795 800 Lys Tyr Ala MetPhe Tyr Pro Arg Asn 805 109 18 PRT Homo sapiens 109 Leu Lys Ile Ile IleSer Ile Ile Leu Pro Pro Thr Ile Leu Thr Leu 1 5 10 15 Glu Phe 110 18 PRTHomo sapiens 110 Ile Val Lys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala PheLeu Met 1 5 10 15 Leu Phe 111 13 PRT Homo sapiens 111 Thr Glu Thr ValAla Ile Gly Leu Phe Ser Ala Gly Phe 1 5 10 112 18 PRT Homo sapiens 112Arg Leu Ile Tyr Cys Ile Asp Ile Ile Phe Trp Phe Ser Arg Leu Leu 1 5 1015 Asp Phe 113 18 PRT Homo sapiens 113 Met Thr Ala Asn Met Phe Tyr IleVal Ile Ile Met Ala Ile Val Leu 1 5 10 15 Leu Ser 114 20 PRT Homosapiens 114 Phe Leu Gln Ala Val Tyr Leu Phe Val Gln Tyr Ile Ile Met ValAsn 1 5 10 15 Leu Leu Ile Ala 20 115 13 PRT Homo sapiens 115 Ile Ile LeuSer Lys Ser Gln Lys Ser Trp Ile Lys Gly 1 5 10 116 13 PRT Homo sapiens116 Ser Thr Ile Ile Pro Ser Ser Lys Asn Pro His Arg Cys 1 5 10 117 13PRT Homo sapiens 117 Ser Val Glu Lys His Thr Thr Lys Ser Pro Thr Asp Thr1 5 10 118 13 PRT Homo sapiens 118 Ser His Ser Ser His Ser Leu Arg LysIle Trp Thr Val 1 5 10 119 13 PRT Homo sapiens 119 Leu Ser Val Trp GluThr Val Lys Asp Lys Asp Pro Val 1 5 10 120 13 PRT Homo sapiens 120 ValVal Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu Leu 1 5 10 121 13 PRT Homosapiens 121 Asp Leu Leu Ala Phe Thr His Lys His Leu Ala Asp Glu 1 5 10122 13 PRT Homo sapiens 122 Asn Thr Phe Asn Phe Ser Leu Lys Gln Ser LysHis Leu 1 5 10 123 13 PRT Homo sapiens 123 Ile Val Leu His Lys Ser ArgLys Lys Ser Lys Glu Gln 1 5 10 124 13 PRT Homo sapiens 124 His Gly GluGlu Ala Thr Val Lys Ala Val Ile Ala Cys 1 5 10 125 13 PRT Homo sapiens125 Asp Gln Asn Ala Ser Ser Ser Lys Glu Ser Ala Ser Val 1 5 10 126 13PRT Homo sapiens 126 Ser Lys Glu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu1 5 10 127 13 PRT Homo sapiens 127 Gln His Leu Pro Trp Thr Arg Lys ValTyr Glu Phe Tyr 1 5 10 128 13 PRT Homo sapiens 128 Glu Pro Gly Lys PheThr Gln Lys Val Lys Val Trp Ile 1 5 10 129 13 PRT Homo sapiens 129 ArgLys Ala Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp 1 5 10 130 13 PRT Homosapiens 130 Arg Ile Arg Val Thr Ser Glu Arg Val Thr Glu Met Tyr 1 5 10131 13 PRT Homo sapiens 131 Ala Leu Thr Val Asp Thr Leu Lys Val Leu SerAla Val 1 5 10 132 13 PRT Homo sapiens 132 Lys Arg Lys His Ser Thr CysLys Lys Leu Pro His Ser 1 5 10 133 13 PRT Homo sapiens 133 Leu Glu IleThr Asn Ser Lys Arg Glu Ala Thr Asn Val 1 5 10 134 13 PRT Homo sapiens134 Glu Thr Gly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr 1 5 10 135 13PRT Homo sapiens 135 Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln1 5 10 136 13 PRT Homo sapiens 136 Ser Leu Ser Asp Asn Ser Thr Arg SerAla Gln Ser Ser 1 5 10 137 13 PRT Homo sapiens 137 Phe Ala Arg Ser HisSer Phe Arg Phe His Lys Glu Glu 1 5 10 138 13 PRT Homo sapiens 138 LysAsp Arg Arg Leu Ser Lys Lys Lys Lys Asn Thr Gln 1 5 10 139 13 PRT Homosapiens 139 Asp Lys Ile Ser Ala Ser Leu Lys Ser Pro Gln Glu Pro 1 5 10140 13 PRT Homo sapiens 140 Ser Met Ser Ser Trp Ser Gln Arg Gly Arg AlaAla Met 1 5 10 141 13 PRT Homo sapiens 141 Gln Thr Ile Pro Tyr Thr ProArg Phe Leu Glu Val Phe 1 5 10 142 13 PRT Homo sapiens 142 Pro Pro AlaArg Glu Thr Gly Arg Asn Ser Pro Glu Asp 1 5 10 143 14 PRT Homo sapiens143 His Gly Gly Ile Gln Asn Phe Thr Met Pro Ser Lys Phe Lys 1 5 10 14414 PRT Homo sapiens 144 Ile Gln Asn Thr Phe Asn Phe Ser Leu Lys Gln SerLys His 1 5 10 145 14 PRT Homo sapiens 145 Leu Leu Lys Gly Thr Asn LeuSer Ala Ser Glu Gln Leu Asn 1 5 10 146 14 PRT Homo sapiens 146 Ser SerGly Asn Arg Asn Glu Ser Ala Glu Ser Thr Leu His 1 5 10 147 14 PRT Homosapiens 147 Lys Ser Lys Glu Gln Asn Val Ser Asp Asp Pro Glu Ser Thr 1 510 148 14 PRT Homo sapiens 148 Ser Glu Glu Leu Lys Asn Tyr Ser Lys GlnPhe Gly Gln Leu 1 5 10 149 14 PRT Homo sapiens 149 Thr Tyr Glu Leu ArgAsn Trp Ser Asn Ser Thr Cys Leu Lys 1 5 10 150 14 PRT Homo sapiens 150Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys Leu Ala Val 1 5 10 151 14PRT Homo sapiens 151 Tyr Tyr Ser Asp Gln Asn Ala Ser Ser Ser Lys Glu SerAla 1 5 10 152 14 PRT Homo sapiens 152 Ile Ser Glu Tyr Trp Asn Leu ThrGlu Thr Val Ala Ile Gly 1 5 10 153 14 PRT Homo sapiens 153 Lys Met GluAsp Val Asn Cys Ser Cys Glu Glu Arg Ile Arg 1 5 10 154 14 PRT Homosapiens 154 Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln Ser Ser 1 510 155 14 PRT Homo sapiens 155 Pro Trp Leu Gln Pro Asn Thr Ser Phe TrpIle Asn Pro Leu 1 5 10 156 14 PRT Homo sapiens 156 Ile Cys Lys Ile LysAsn Leu Ser Gly Ser Ser Glu Ile Gly 1 5 10 157 14 PRT Homo sapiens 157Gln Gly Val Gly Glu Asn Leu Thr Asp Pro Ser Val Ile Lys 1 5 10 158 14PRT Homo sapiens 158 Ser Pro Glu Arg Ile Asn Ser Thr Phe Gly Leu Glu IleLys 1 5 10 159 18 PRT Homo sapiens 159 Leu Lys Ile Ile Ile Ser Ile IleLeu Pro Pro Thr Ile Leu Thr Leu 1 5 10 15 Glu Phe 160 18 PRT Homosapiens 160 Ile Val Lys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala Phe LeuMet 1 5 10 15 Leu Phe 161 13 PRT Homo sapiens 161 Thr Glu Thr Val AlaIle Gly Leu Phe Ser Ala Gly Phe 1 5 10 162 18 PRT Homo sapiens 162 ArgLeu Ile Tyr Cys Ile Asp Ile Ile Phe Trp Phe Ser Arg Leu Leu 1 5 10 15Asp Phe 163 18 PRT Homo sapiens 163 Met Thr Ala Asn Met Phe Tyr Ile ValIle Ile Met Ala Ile Val Leu 1 5 10 15 Leu Ser 164 20 PRT Homo sapiens164 Phe Leu Gln Ala Val Tyr Leu Phe Val Gln Tyr Ile Ile Met Val Asn 1 510 15 Leu Leu Ile Ala 20 165 13 PRT Homo sapiens 165 Ile Ile Leu Ser LysSer Gln Lys Ser Trp Ile Lys Gly 1 5 10 166 13 PRT Homo sapiens 166 SerThr Ile Ile Pro Ser Ser Lys Asn Pro His Arg Cys 1 5 10 167 13 PRT Homosapiens 167 Ser Val Glu Lys His Thr Thr Lys Ser Pro Thr Asp Thr 1 5 10168 13 PRT Homo sapiens 168 Ser His Ser Ser His Ser Leu Arg Lys Ile TrpThr Val 1 5 10 169 13 PRT Homo sapiens 169 Leu Ser Val Trp Glu Thr ValLys Asp Lys Asp Pro Val 1 5 10 170 13 PRT Homo sapiens 170 Val Val CysGlu Gly Thr Gly Arg Ala Ala Asp Leu Leu 1 5 10 171 13 PRT Homo sapiens171 Asp Leu Leu Ala Phe Thr His Lys His Leu Ala Asp Glu 1 5 10 172 13PRT Homo sapiens 172 Asn Thr Phe Asn Phe Ser Leu Lys Gln Ser Lys His Leu1 5 10 173 13 PRT Homo sapiens 173 Ile Val Leu His Lys Ser Arg Lys LysSer Lys Glu Gln 1 5 10 174 13 PRT Homo sapiens 174 His Gly Glu Glu AlaThr Val Lys Ala Val Ile Ala Cys 1 5 10 175 13 PRT Homo sapiens 175 AspGln Asn Ala Ser Ser Ser Lys Glu Ser Ala Ser Val 1 5 10 176 13 PRT Homosapiens 176 Ser Lys Glu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu 1 5 10177 13 PRT Homo sapiens 177 Gln His Leu Pro Trp Thr Arg Lys Val Tyr GluPhe Tyr 1 5 10 178 13 PRT Homo sapiens 178 Glu Pro Gly Lys Phe Thr GlnLys Val Lys Val Trp Ile 1 5 10 179 13 PRT Homo sapiens 179 Arg Lys AlaIle Leu Ser Pro Lys Glu Pro Pro Ser Trp 1 5 10 180 13 PRT Homo sapiens180 Arg Ile Arg Val Thr Ser Glu Arg Val Thr Glu Met Tyr 1 5 10 181 13PRT Homo sapiens 181 Ala Leu Thr Val Asp Thr Leu Lys Val Leu Ser Ala Val1 5 10 182 13 PRT Homo sapiens 182 Lys Arg Lys His Ser Thr Cys Lys LysLeu Pro His Ser 1 5 10 183 13 PRT Homo sapiens 183 Leu Glu Ile Thr AsnSer Lys Arg Glu Ala Thr Asn Val 1 5 10 184 13 PRT Homo sapiens 184 GluThr Gly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr 1 5 10 185 13 PRT Homosapiens 185 Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln 1 5 10186 13 PRT Homo sapiens 186 Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala GlnSer Ser 1 5 10 187 13 PRT Homo sapiens 187 Phe Ala Arg Ser His Ser PheArg Phe His Lys Glu Glu 1 5 10 188 13 PRT Homo sapiens 188 Lys Asp ArgArg Leu Ser Lys Lys Lys Lys Asn Thr Gln 1 5 10 189 13 PRT Homo sapiens189 Asp Lys Ile Ser Ala Ser Leu Lys Ser Pro Gln Glu Pro 1 5 10 190 13PRT Homo sapiens 190 Ser Met Ser Ser Trp Ser Gln Arg Gly Arg Ala Ala Met1 5 10 191 13 PRT Homo sapiens 191 Gln Thr Ile Pro Tyr Thr Pro Arg PheLeu Glu Val Phe 1 5 10 192 13 PRT Homo sapiens 192 Pro Pro Ala Arg GluThr Gly Arg Asn Ser Pro Glu Asp 1 5 10 193 14 PRT Homo sapiens 193 HisGly Gly Ile Gln Asn Phe Thr Met Pro Ser Lys Phe Lys 1 5 10 194 14 PRTHomo sapiens 194 Ile Gln Asn Thr Phe Asn Phe Ser Leu Lys Gln Ser Lys His1 5 10 195 14 PRT Homo sapiens 195 Leu Leu Lys Gly Thr Asn Leu Ser AlaSer Glu Gln Leu Asn 1 5 10 196 14 PRT Homo sapiens 196 Lys Ser Lys GluGln Asn Val Ser Asp Asp Pro Glu Ser Thr 1 5 10 197 14 PRT Homo sapiens197 Ser Glu Glu Leu Lys Asn Tyr Ser Lys Gln Phe Gly Gln Leu 1 5 10 19814 PRT Homo sapiens 198 Thr Tyr Glu Leu Arg Asn Trp Ser Asn Ser Thr CysLeu Lys 1 5 10 199 14 PRT Homo sapiens 199 Leu Arg Asn Trp Ser Asn SerThr Cys Leu Lys Leu Ala Val 1 5 10 200 14 PRT Homo sapiens 200 Tyr TyrSer Asp Gln Asn Ala Ser Ser Ser Lys Glu Ser Ala 1 5 10 201 14 PRT Homosapiens 201 Ile Ser Glu Tyr Trp Asn Leu Thr Glu Thr Val Ala Ile Gly 1 510 202 14 PRT Homo sapiens 202 Lys Met Glu Asp Val Asn Cys Ser Cys GluGlu Arg Ile Arg 1 5 10 203 14 PRT Homo sapiens 203 Ser Ser Leu Ser AspAsn Ser Thr Arg Ser Ala Gln Ser Ser 1 5 10 204 14 PRT Homo sapiens 204Pro Trp Leu Gln Pro Asn Thr Ser Phe Trp Ile Asn Pro Leu 1 5 10 205 14PRT Homo sapiens 205 Ile Cys Lys Ile Lys Asn Leu Ser Gly Ser Ser Glu IleGly 1 5 10 206 14 PRT Homo sapiens 206 Gln Gly Val Gly Glu Asn Leu ThrAsp Pro Ser Val Ile Lys 1 5 10 207 14 PRT Homo sapiens 207 Ser Pro GluArg Ile Asn Ser Thr Phe Gly Leu Glu Ile Lys 1 5 10 208 18 PRT Homosapiens 208 Leu Lys Ile Ile Ile Ser Ile Ile Leu Pro Pro Thr Ile Leu ThrLeu 1 5 10 15 Glu Phe 209 12 PRT Homo sapiens 209 Ile Val Lys Phe TrpPhe Tyr Thr Ile Cys Ile Ser 1 5 10 210 20 PRT Homo sapiens 210 Tyr TrpAsn Leu Thr Glu Thr Val Ala Ile Gly Leu Phe Ser Ala Gly 1 5 10 15 PheVal Leu Arg 20 211 18 PRT Homo sapiens 211 Leu Ile Tyr Cys Ile Asp IleIle Phe Trp Phe Ser Arg Leu Leu Asp 1 5 10 15 Phe Phe 212 22 PRT Homosapiens 212 Met Thr Ala Asn Met Phe Tyr Ile Val Ile Ile Met Ala Ile ValLeu 1 5 10 15 Leu Ser Phe Gly Val Ala 20 213 31 PRT Homo sapiens 213 PheLeu Thr Pro Phe Leu Gln Ala Val Tyr Leu Phe Val Gln Tyr Ile 1 5 10 15Ile Met Val Asn Leu Leu Ile Ala Phe Phe Asn Asn Val Tyr Leu 20 25 30 21413 PRT Homo sapiens 214 Ile Ile Leu Ser Lys Ser Gln Lys Ser Trp Ile LysGly 1 5 10 215 13 PRT Homo sapiens 215 Ser Thr Ile Ile Pro Ser Ser LysAsn Pro His Arg Cys 1 5 10 216 13 PRT Homo sapiens 216 Ser Val Glu LysHis Thr Thr Lys Ser Pro Thr Asp Thr 1 5 10 217 13 PRT Homo sapiens 217Ser His Ser Ser His Ser Leu Arg Lys Ile Trp Thr Val 1 5 10 218 13 PRTHomo sapiens 218 Leu Ser Val Trp Glu Thr Val Lys Asp Lys Asp Pro Val 1 510 219 13 PRT Homo sapiens 219 Val Val Cys Glu Gly Thr Gly Arg Ala AlaAsp Leu Leu 1 5 10 220 13 PRT Homo sapiens 220 Asp Leu Leu Ala Phe ThrHis Lys His Leu Ala Asp Glu 1 5 10 221 13 PRT Homo sapiens 221 Asn ThrPhe Asn Phe Ser Leu Lys Gln Ser Lys His Leu 1 5 10 222 13 PRT Homosapiens 222 Tyr Arg Ser Asn Tyr Thr Arg Lys His Phe Arg Ala Leu 1 5 10223 13 PRT Homo sapiens 223 Ile Val Leu His Lys Ser Arg Lys Lys Ser LysGlu Gln 1 5 10 224 13 PRT Homo sapiens 224 His Gly Glu Glu Ala Thr ValLys Ala Val Ile Ala Cys 1 5 10 225 13 PRT Homo sapiens 225 Asp Gln AsnAla Ser Ser Ser Lys Glu Ser Ala Ser Val 1 5 10 226 13 PRT Homo sapiens226 Ser Lys Glu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu 1 5 10 227 13PRT Homo sapiens 227 Gln His Leu Pro Trp Thr Arg Lys Val Tyr Glu Phe Tyr1 5 10 228 13 PRT Homo sapiens 228 Glu Pro Gly Lys Phe Thr Gln Lys ValLys Val Trp Ile 1 5 10 229 13 PRT Homo sapiens 229 Arg Lys Ala Ile LeuSer Pro Lys Glu Pro Pro Ser Trp 1 5 10 230 13 PRT Homo sapiens 230 ArgIle Arg Val Thr Ser Glu Arg Val Thr Glu Met Tyr 1 5 10 231 13 PRT Homosapiens 231 Ala Leu Thr Val Asp Thr Leu Lys Val Leu Ser Ala Val 1 5 10232 13 PRT Homo sapiens 232 Lys Arg Lys His Ser Thr Cys Lys Lys Leu ProHis Ser 1 5 10 233 13 PRT Homo sapiens 233 Leu Glu Ile Thr Asn Ser LysArg Glu Ala Thr Asn Val 1 5 10 234 13 PRT Homo sapiens 234 Glu Thr GlyVal Phe Ser Ile Lys Lys Lys Trp Gln Thr 1 5 10 235 13 PRT Homo sapiens235 Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln 1 5 10 236 13PRT Homo sapiens 236 Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln Ser Ser1 5 10 237 13 PRT Homo sapiens 237 Phe Ala Arg Ser His Ser Phe Arg PheHis Lys Glu Glu 1 5 10 238 13 PRT Homo sapiens 238 Lys Asp Arg Arg LeuSer Lys Lys Lys Lys Asn Thr Gln 1 5 10 239 13 PRT Homo sapiens 239 AspLys Ile Ser Ala Ser Leu Lys Ser Pro Gln Glu Pro 1 5 10 240 13 PRT Homosapiens 240 Ser Met Ser Ser Trp Ser Gln Arg Gly Arg Ala Ala Met 1 5 10241 13 PRT Homo sapiens 241 Gln Thr Ile Pro Tyr Thr Pro Arg Phe Leu GluVal Phe 1 5 10 242 13 PRT Homo sapiens 242 Pro Pro Ala Arg Glu Thr GlyArg Asn Ser Pro Glu Asp 1 5 10 243 14 PRT Homo sapiens 243 His Gly GlyIle Gln Asn Phe Thr Met Pro Ser Lys Phe Lys 1 5 10 244 14 PRT Homosapiens 244 Ile Gln Asn Thr Phe Asn Phe Ser Leu Lys Gln Ser Lys His 1 510 245 14 PRT Homo sapiens 245 Leu Leu Lys Gly Thr Asn Leu Ser Ala SerGlu Gln Leu Asn 1 5 10 246 14 PRT Homo sapiens 246 Arg Ala Tyr Arg SerAsn Tyr Thr Arg Lys His Phe Arg Ala 1 5 10 247 14 PRT Homo sapiens 247Ser Ser Gly Asn Arg Asn Glu Ser Ala Glu Ser Thr Leu His 1 5 10 248 14PRT Homo sapiens 248 Lys Ser Lys Glu Gln Asn Val Ser Asp Asp Pro Glu SerThr 1 5 10 249 14 PRT Homo sapiens 249 Ser Glu Glu Leu Lys Asn Tyr SerLys Gln Phe Gly Gln Leu 1 5 10 250 14 PRT Homo sapiens 250 Thr Tyr GluLeu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys 1 5 10 251 14 PRT Homosapiens 251 Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys Leu Ala Val 1 510 252 14 PRT Homo sapiens 252 Tyr Tyr Ser Asp Gln Asn Ala Ser Ser SerLys Glu Ser Ala 1 5 10 253 14 PRT Homo sapiens 253 Ile Ser Glu Tyr TrpAsn Leu Thr Glu Thr Val Ala Ile Gly 1 5 10 254 14 PRT Homo sapiens 254Lys Met Glu Asp Val Asn Cys Ser Cys Glu Glu Arg Ile Arg 1 5 10 255 14PRT Homo sapiens 255 Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln SerSer 1 5 10 256 14 PRT Homo sapiens 256 Pro Trp Leu Gln Pro Asn Thr SerPhe Trp Ile Asn Pro Leu 1 5 10 257 14 PRT Homo sapiens 257 Ile Cys LysIle Lys Asn Leu Ser Gly Ser Ser Glu Ile Gly 1 5 10 258 14 PRT Homosapiens 258 Gln Gly Val Gly Glu Asn Leu Thr Asp Pro Ser Val Ile Lys 1 510 259 14 PRT Homo sapiens 259 Ser Pro Glu Arg Ile Asn Ser Thr Phe GlyLeu Glu Ile Lys 1 5 10 260 1533 PRT Homo sapiens 260 Met Tyr Ile Arg ValSer Tyr Asp Thr Lys Pro Asp Ser Leu Leu His 1 5 10 15 Leu Met Val LysAsp Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val His Gly GlyLeu Gln Asn Phe Glu Met Gln Pro Lys Leu Lys Gln 35 40 45 Val Phe Gly LysGly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 Ile Phe Thr GlyGly Val Ser Thr Gly Val Ile Ser His Val Gly Asp 65 70 75 80 Ala Leu LysAsp His Ser Ser Lys Ser Arg Gly Arg Val Cys Ala Ile 85 90 95 Gly Ile AlaPro Trp Gly Ile Val Glu Asn Lys Glu Asp Leu Val Gly 100 105 110 Lys AspVal Thr Arg Val Tyr Gln Thr Met Ser Asn Pro Leu Ser Lys 115 120 125 LeuSer Val Leu Asn Asn Ser His Thr His Phe Ile Leu Ala Asp Asn 130 135 140Gly Thr Leu Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Leu Leu 145 150155 160 Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly165 170 175 Val Pro Leu Val Gly Leu Val Val Glu Gly Gly Pro Asn Val ValSer 180 185 190 Ile Val Leu Glu Tyr Leu Gln Glu Glu Pro Pro Ile Pro ValVal Ile 195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu Ser PheAla His Lys 210 215 220 Tyr Cys Glu Glu Gly Gly Ile Ile Asn Glu Ser LeuArg Glu Gln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys Thr Phe Asn TyrAsn Lys Ala Gln Ser His 245 250 255 Gln Leu Phe Ala Ile Ile Met Glu CysMet Lys Lys Lys Glu Leu Val 260 265 270 Thr Val Phe Arg Met Gly Ser GluGly Gln Gln Asp Ile Glu Met Ala 275 280 285 Ile Leu Thr Ala Leu Leu LysGly Thr Asn Val Ser Ala Pro Asp Gln 290 295 300 Leu Ser Leu Ala Leu AlaTrp Asn Arg Val Asp Ile Ala Arg Ser Gln 305 310 315 320 Ile Phe Val PheGly Pro His Trp Thr Pro Leu Gly Ser Leu Ala Pro 325 330 335 Pro Thr AspSer Lys Ala Thr Glu Lys Glu Lys Lys Pro Pro Met Ala 340 345 350 Thr ThrLys Gly Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Gly Lys 355 360 365 ValLys Glu Glu Val Glu Glu Glu Thr Asp Pro Arg Lys Ile Glu Leu 370 375 380Leu Asn Trp Val Asn Ala Leu Glu Gln Ala Met Leu Asp Ala Leu Val 385 390395 400 Leu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val Asn405 410 415 Met Gln His Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr AsnThr 420 425 430 Arg Leu Gly Pro Pro Asn Thr Leu His Leu Leu Val Arg AspVal Lys 435 440 445 Lys Ser Asn Leu Pro Pro Asp Tyr His Ile Ser Leu IleAsp Ile Gly 450 455 460 Leu Val Leu Glu Tyr Leu Met Gly Gly Ala Tyr ArgCys Asn Tyr Thr 465 470 475 480 Arg Lys Asn Phe Arg Thr Leu Tyr Asn AsnLeu Phe Gly Pro Lys Arg 485 490 495 Pro Lys Ala Leu Lys Leu Leu Gly MetGlu Asp Asp Glu Pro Pro Ala 500 505 510 Lys Gly Lys Lys Lys Lys Lys LysLys Lys Glu Glu Glu Ile Asp Ile 515 520 525 Asp Val Asp Asp Pro Ala ValSer Arg Phe Gln Tyr Pro Phe His Glu 530 535 540 Leu Met Val Trp Ala ValLeu Met Lys Arg Gln Lys Met Ala Val Phe 545 550 555 560 Leu Trp Gln ArgGly Glu Glu Ser Met Ala Lys Ala Leu Val Ala Cys 565 570 575 Lys Leu TyrLys Ala Met Ala His Glu Ser Ser Glu Ser Asp Leu Val 580 585 590 Asp AspIle Ser Gln Asp Leu Asp Asn Asn Ser Lys Asp Phe Gly Gln 595 600 605 LeuAla Leu Glu Leu Leu Asp Gln Ser Tyr Lys His Asp Glu Gln Ile 610 615 620Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ser Thr 625 630635 640 Cys Leu Lys Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His645 650 655 Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg LeuArg 660 665 670 Met Arg Lys Asn Pro Gly Leu Lys Val Ile Met Gly Ile LeuLeu Pro 675 680 685 Pro Thr Ile Leu Phe Leu Glu Phe Arg Thr Tyr Asp AspPhe Ser Tyr 690 695 700 Gln Thr Ser Lys Glu Asn Glu Asp Gly Lys Glu LysGlu Glu Glu Asn 705 710 715 720 Thr Asp Ala Asn Ala Asp Ala Gly Ser ArgLys Gly Asp Glu Glu Asn 725 730 735 Glu His Lys Lys Gln Arg Ser Ile ProIle Gly Thr Lys Ile Cys Glu 740 745 750 Phe Tyr Asn Ala Pro Ile Val LysPhe Trp Phe Tyr Thr Ile Ser Tyr 755 760 765 Leu Gly Tyr Leu Leu Leu PheAsn Tyr Val Ile Leu Val Arg Met Asp 770 775 780 Gly Trp Pro Ser Leu GlnGlu Trp Ile Val Ile Ser Tyr Ile Val Ser 785 790 795 800 Leu Ala Leu GluLys Ile Arg Glu Ile Leu Met Ser Glu Pro Gly Lys 805 810 815 Leu Ser GlnLys Ile Lys Val Trp Leu Gln Glu Tyr Trp Asn Ile Thr 820 825 830 Asp LeuVal Ala Ile Ser Thr Phe Met Ile Gly Ala Ile Leu Arg Leu 835 840 845 GlnAsn Gln Pro Tyr Met Gly Tyr Gly Arg Val Ile Tyr Cys Val Asp 850 855 860Ile Ile Phe Trp Tyr Ile Arg Val Leu Asp Ile Phe Gly Val Asn Lys 865 870875 880 Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met885 890 895 Leu Tyr Phe Val Val Ile Met Leu Val Val Leu Met Ser Phe GlyVal 900 905 910 Ala Arg Gln Ala Ile Leu His Pro Glu Glu Lys Pro Ser TrpLys Leu 915 920 925 Ala Arg Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile TyrGly Glu Val 930 935 940 Phe Ala Asp Gln Ile Asp Leu Tyr Ala Met Glu IleAsn Pro Pro Cys 945 950 955 960 Gly Glu Asn Leu Tyr Asp Glu Glu Gly LysArg Leu Pro Pro Cys Ile 965 970 975 Pro Gly Ala Trp Leu Thr Pro Ala LeuMet Ala Cys Tyr Leu Leu Val 980 985 990 Ala Asn Ile Leu Leu Val Asn LeuLeu Ile Ala Val Phe Asn Asn Thr 995 1000 1005 Phe Phe Glu Val Lys SerIle Ser Asn Gln Val Trp Lys Phe Gln 1010 1015 1020 Arg Tyr Gln Leu IleMet Thr Phe His Asp Arg Pro Val Leu Pro 1025 1030 1035 Pro Pro Met IleIle Leu Ser His Ile Tyr Ile Ile Ile Met Arg 1040 1045 1050 Leu Ser GlyArg Cys Arg Lys Lys Arg Glu Gly Asp Gln Glu Glu 1055 1060 1065 Arg AspArg Gly Leu Lys Leu Phe Leu Ser Asp Glu Glu Leu Lys 1070 1075 1080 ArgLeu His Glu Phe Glu Glu Gln Cys Val Gln Glu His Phe Arg 1085 1090 1095Glu Lys Glu Asp Glu Gln Gln Ser Ser Ser Asp Glu Arg Ile Arg 1100 11051110 Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu 11151120 1125 Ile Asn Glu Arg Glu Thr Phe Met Lys Thr Ser Leu Gln Thr Val1130 1135 1140 Asp Leu Arg Leu Ala Gln Leu Glu Glu Leu Ser Asn Arg MetVal 1145 1150 1155 Asn Ala Leu Glu Asn Leu Ala Gly Ile Asp Arg Ser AspLeu Ile 1160 1165 1170 Gln Ala Arg Ser Arg Ala Ser Ser Glu Cys Glu AlaThr Tyr Leu 1175 1180 1185 Leu Arg Gln Ser Ser Ile Asn Ser Ala Asp GlyTyr Ser Leu Tyr 1190 1195 1200 Arg Tyr His Phe Asn Gly Glu Glu Leu LeuPhe Glu Asp Thr Ser 1205 1210 1215 Leu Ser Thr Ser Pro Gly Thr Gly ValArg Lys Lys Thr Cys Ser 1220 1225 1230 Phe Arg Ile Lys Glu Glu Lys AspVal Lys Thr His Leu Val Pro 1235 1240 1245 Glu Cys Gln Asn Ser Leu HisLeu Ser Leu Gly Thr Ser Thr Ser 1250 1255 1260 Ala Thr Pro Asp Gly SerHis Leu Ala Val Asp Asp Leu Lys Asn 1265 1270 1275 Ala Glu Glu Ser LysLeu Gly Pro Asp Ile Gly Ile Ser Lys Glu 1280 1285 1290 Asp Asp Glu ArgGln Thr Asp Ser Lys Lys Glu Glu Thr Ile Ser 1295 1300 1305 Pro Ser LeuAsn Lys Thr Asp Val Ile His Gly Gln Asp Lys Ser 1310 1315 1320 Asp ValGln Asn Thr Gln Leu Thr Val Glu Thr Thr Asn Ile Glu 1325 1330 1335 GlyThr Ile Ser Tyr Pro Leu Glu Glu Thr Lys Ile Thr Arg Tyr 1340 1345 1350Phe Pro Asp Glu Thr Ile Asn Ala Cys Lys Thr Met Lys Ser Arg 1355 13601365 Ser Phe Val Tyr Ser Arg Gly Arg Lys Leu Val Gly Gly Val Asn 13701375 1380 Gln Asp Val Glu Tyr Ser Ser Ile Thr Asp Gln Gln Leu Thr Thr1385 1390 1395 Glu Trp Gln Cys Gln Val Gln Lys Ile Thr Arg Ser His SerThr 1400 1405 1410 Asp Ile Pro Tyr Ile Val Ser Glu Ala Ala Val Gln AlaGlu Gln 1415 1420 1425 Lys Glu Gln Phe Ala Asp Met Gln Asp Glu His HisVal Ala Glu 1430 1435 1440 Ala Ile Pro Arg Ile Pro Arg Leu Ser Leu ThrIle Thr Asp Arg 1445 1450 1455 Asn Gly Met Glu Asn Leu Leu Ser Val LysPro Asp Gln Thr Leu 1460 1465 1470 Gly Phe Pro Ser Leu Arg Ser Lys SerLeu His Gly His Pro Arg 1475 1480 1485 Asn Val Lys Ser Ile Gln Gly LysLeu Asp Arg Ser Gly His Ala 1490 1495 1500 Ser Ser Val Ser Ser Leu ValIle Val Ser Gly Met Thr Ala Glu 1505 1510 1515 Glu Lys Lys Val Lys LysGlu Lys Ala Ser Thr Glu Thr Glu Cys 1520 1525 1530 261 8 PRTbacteriophage T7 261 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 262 733 DNAhomo sapiens 262 gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgcccagcacctg 60 aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggacaccctcatga 120 tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaagaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagacaaagccgcggg 240 aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctgcaccaggact 300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctcccaacccccatcg 360 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtacaccctgcccc 420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtcaaaggcttct 480 atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaacaactacaaga 540 ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaagctcaccgtgg 600 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcatgaggctctgc 660 acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtgcgacggccgc 720 gactctagag gat 733 263 35 DNA Homo sapiens 263 gcagcagcggccgctggtta aagattatta taagc 35 264 35 DNA Homo sapiens 264 gcagcagtcgactagttgca tatcatcttc tgggg 35 265 38 DNA Homo sapiens 265 gcagcagcggccgcatgatt atcctatcta agtcccag 38 266 37 DNA Homo sapiens 266 gcagcagtcgacgggatgcc ggcctccagc caggctc 37 267 23 DNA Homo sapiens 267 agaaaatacactgccgctca aga 23 268 17 DNA Homo sapiens 268 gttgggaccg ccttcca 17 26919 DNA Homo sapiens 269 cccacgaccg gcacgcctt 19 270 38 DNA Homo sapiens270 cgggatccac gatgggagtt gacaagatct cagcctcc 38 271 67 DNA Homo sapiens271 atagttagcg gccgcttaag cgtaatctgg aacatcgtat gggtatagtt gcatatcatc 60ttctggg 67 272 23 DNA Homo sapiens 272 caggtgcagc tggtgcagtc tgg 23 27323 DNA Homo sapiens 273 caggtcaact taagggagtc tgg 23 274 23 DNA Homosapiens 274 gaggtgcagc tggtggagtc tgg 23 275 23 DNA Homo sapiens 275caggtgcagc tgcaggagtc ggg 23 276 23 DNA Homo sapiens 276 gaggtgcagctgttgcagtc tgc 23 277 23 DNA Homo sapiens 277 caggtacagc tgcagcagtc agg23 278 24 DNA Homo sapiens 278 tgaggagacg gtgaccaggg tgcc 24 279 24 DNAHomo sapiens 279 tgaagagacg gtgaccattg tccc 24 280 24 DNA Homo sapiens280 tgaggagacg gtgaccaggg ttcc 24 281 24 DNA Homo sapiens 281 tgaggagacggtgaccgtgg tccc 24 282 23 DNA Homo sapiens 282 gacatccaga tgacccagtc tcc23 283 23 DNA Homo sapiens 283 gatgttgtga tgactcagtc tcc 23 284 23 DNAHomo sapiens 284 gatattgtga tgactcagtc tcc 23 285 23 DNA Homo sapiens285 gaaattgtgt tgacgcagtc tcc 23 286 23 DNA Homo sapiens 286 gacatcgtgatgacccagtc tcc 23 287 23 DNA Homo sapiens 287 gaaacgacac tcacgcagtc tcc23 288 23 DNA Homo sapiens 288 gaaattgtgc tgactcagtc tcc 23 289 23 DNAHomo sapiens 289 cagtctgtgt tgacgcagcc gcc 23 290 23 DNA Homo sapiens290 cagtctgccc tgactcagcc tgc 23 291 23 DNA Homo sapiens 291 tcctatgtgctgactcagcc acc 23 292 23 DNA Homo sapiens 292 tcttctgagc tgactcagga ccc23 293 23 DNA Homo sapiens 293 cacgttatac tgactcaacc gcc 23 294 23 DNAHomo sapiens 294 caggctgtgc tcactcagcc gtc 23 295 23 DNA Homo sapiens295 aattttatgc tgactcagcc cca 23 296 24 DNA Homo sapiens 296 acgtttgatttccaccttgg tccc 24 297 24 DNA Homo sapiens 297 acgtttgatc tccagcttggtccc 24 298 24 DNA Homo sapiens 298 acgtttgata tccactttgg tccc 24 299 24DNA Homo sapiens 299 acgtttgatc tccaccttgg tccc 24 300 24 DNA Homosapiens 300 acgtttaatc tccagtcgtg tccc 24 301 23 DNA Homo sapiens 301cagtctgtgt tgacgcagcc gcc 23 302 23 DNA Homo sapiens 302 cagtctgccctgactcagcc tgc 23 303 23 DNA Homo sapiens 303 tcctatgtgc tgactcagcc acc23 304 23 DNA Homo sapiens 304 tcttctgagc tgactcagga ccc 23 305 23 DNAHomo sapiens 305 cacgttatac tgactcaacc gcc 23 306 23 DNA Homo sapiens306 caggctgtgc tcactcagcc gtc 23 307 23 DNA Homo sapiens 307 aattttatgctgactcagcc cca 23 308 24 DNA Homo sapiens 308 gaggatgagg agagctatga caca24 309 22 DNA Homo sapiens 309 ccctttgcac tcataacgtc ag 22 310 29 DNAHomo sapiens 310 aaacacacag tcatcatagg gcagctcgt 29 311 25 DNAArtificial Sequence Synthesized Oligonucleotide 311 ccuugacagucucccacacu gacag 25 312 204 PRT Homo sapiens 312 Val Lys Asp Lys Asp ProVal Val Val Cys Glu Gly Thr Gly Arg Ala 1 5 10 15 Ala Asp Leu Leu AlaPhe Thr His Lys His Leu Ala Asp Glu Gly Met 20 25 30 Leu Arg Pro Gln ValLys Glu Glu Ile Ile Cys Met Ile Gln Asn Thr 35 40 45 Phe Asn Phe Ser LeuLys Gln Ser Lys His Leu Phe Gln Ile Leu Met 50 55 60 Glu Cys Met Val HisArg Asp Cys Ile Thr Ile Phe Asp Ala Asp Ser 65 70 75 80 Glu Glu Gln GlnAsp Leu Asp Leu Ala Ile Leu Thr Ala Leu Leu Lys 85 90 95 Gly Thr Asn LeuSer Ala Ser Glu Gln Leu Asn Leu Ala Met Ala Trp 100 105 110 Asp Arg ValAsp Ile Ala Lys Lys His Ile Leu Ile Tyr Glu Gln His 115 120 125 Trp LysPro Asp Ala Leu Glu Gln Ala Met Ser Asp Ala Leu Val Met 130 135 140 AspArg Val Asp Phe Val Lys Leu Leu Ile Glu Tyr Gly Val Asn Leu 145 150 155160 His Arg Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr Lys 165170 175 Gln Gly Pro Thr Asn Thr Leu Leu His His Leu Val Gln Asp Val Lys180 185 190 Gln His Thr Leu Leu Ser Gly Tyr Arg Ile Thr Leu 195 200 31317 PRT Homo sapiens 313 Lys Ser Pro Thr Asp Thr Phe Gly Thr Ile Asn PheGln Asp Gly Glu 1 5 10 15 His 314 24 PRT Homo sapiens 314 Asp His LeuLeu His Leu Met Leu Lys Glu Trp Met Glu Leu Pro Lys 1 5 10 15 Leu ValIle Ser Val His Gly Gly 20 315 22 PRT Homo sapiens 315 Phe Ser Gln GlyLeu Val Lys Ala Ala Glu Thr Thr Gly Ala Trp Ile 1 5 10 15 Ile Thr GluGly Ile Asn 20 316 20 PRT Homo sapiens 316 Ser Leu Arg Lys Ile Trp ThrVal Gly Ile Pro Pro Trp Gly Val Ile 1 5 10 15 Glu Asn Gln Arg 20 317 19PRT Homo sapiens 317 Thr Val Leu His Leu Cys Leu Arg Glu Ile Gln Gln GlnArg Ala Ala 1 5 10 15 Gln Lys Leu 318 16 PRT Homo sapiens 318 Met ThrGly Glu Phe Arg Lys Tyr Asn Asn Asn Asn Gly Asp Glu Ile 1 5 10 15 319 35PRT Homo sapiens 319 Met Leu Ala Phe Ser His Trp Thr Tyr Glu Tyr Thr ArgGly Glu Leu 1 5 10 15 Leu Val Leu Asp Leu Gln Gly Val Gly Glu Asn LeuThr Asp Pro Ser 20 25 30 Val Ile Lys 35 320 22 PRT Homo sapiens 320 AlaLys His His Cys Asn Ser Cys Cys Arg Lys Leu Lys Leu Pro Asp 1 5 10 15Leu Lys Arg Asn Asp Tyr 20 321 158 PRT Homo sapiens 321 Gly Lys Asp ValVal Cys Leu Tyr Gln Thr Leu Asp Asn Pro Leu Ser 1 5 10 15 Lys Leu ThrThr Leu Asn Ser Met His Ser His Phe Ile Leu Ser Asp 20 25 30 Asp Gly ThrVal Gly Lys Tyr Gly Asn Glu Met Lys Leu Arg Arg Asn 35 40 45 Leu Glu LysTyr Leu Ser Leu Gln Lys Ile His Cys Arg Ser Arg Gln 50 55 60 Gly Val ProVal Val Gly Leu Val Val Glu Gly Gly Pro Asn Val Ile 65 70 75 80 Leu SerVal Trp Glu Thr Val Lys Asp Lys Asp Pro Val Val Val Cys 85 90 95 Glu GlyThr Gly Arg Ala Ala Asp Leu Leu Ala Phe Thr His Lys His 100 105 110 LeuAla Asp Glu Gly Met Leu Arg Pro Gln Val Lys Glu Glu Ile Ile 115 120 125Cys Met Ile Gln Asn Thr Phe Asn Phe Ser Leu Lys Gln Ser Lys His 130 135140 Leu Phe Gln Ile Leu Met Glu Cys Met Val His Arg Asp Cys 145 150 155322 26 PRT Homo sapiens 322 Glu Tyr Thr Arg Gly Glu Leu Leu Val Leu AspLeu Gln Gly Val Gly 1 5 10 15 Glu Asn Leu Thr Asp Pro Ser Val Ile Lys 2025 323 16 PRT Homo sapiens 323 Tyr Pro Tyr Asn Asp Leu Leu Val Trp AlaVal Leu Met Lys Arg Gln 1 5 10 15 324 19 PRT Homo sapiens 324 Met AlaMet Phe Phe Trp Gln His Gly Glu Glu Ala Thr Val Lys Ala 1 5 10 15 ValIle Ala 325 22 PRT Homo sapiens 325 Asn Trp Ser Asn Ser Thr Cys Leu LysLeu Ala Val Ser Gly Gly Leu 1 5 10 15 Arg Pro Phe Val Ser His 20 326 26PRT Homo sapiens 326 Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu LysMet Arg Lys 1 5 10 15 Asn Ser Trp Leu Lys Ile Ile Ile Ser Ile 20 25 32717 PRT Homo sapiens 327 Leu Lys Pro Gly Gln Val Phe Ile Val Lys Ser PheLeu Pro Glu Val 1 5 10 15 Val 328 46 PRT Homo sapiens 328 Lys Ile PheGln Glu Ser Thr Val Leu His Leu Cys Leu Arg Glu Ile 1 5 10 15 Gln GlnGln Arg Ala Ala Gln Lys Leu Ile Tyr Thr Phe Asn Gln Val 20 25 30 Lys ProGln Thr Ile Pro Tyr Thr Pro Arg Phe Leu Glu Val 35 40 45 329 30 PRT Homosapiens 329 Tyr Cys His Ser Ala Asn Gln Trp Leu Thr Ile Glu Lys Tyr MetThr 1 5 10 15 Gly Glu Phe Arg Lys Tyr Asn Asn Asn Asn Gly Asp Glu Ile 2025 30 330 39 PRT Homo sapiens 330 Pro Thr Asn Thr Leu Glu Glu Leu MetLeu Ala Phe Ser His Trp Thr 1 5 10 15 Tyr Glu Tyr Thr Arg Gly Glu LeuLeu Val Leu Asp Leu Gln Gly Val 20 25 30 Gly Glu Asn Leu Thr Asp Pro 35331 19 PRT Homo sapiens 331 Thr Val Leu His Leu Cys Leu Arg Glu Ile GlnGln Gln Arg Ala Ala 1 5 10 15 Gln Lys Leu 332 16 PRT Homo sapiens 332Met Thr Gly Glu Phe Arg Lys Tyr Asn Asn Asn Asn Gly Asp Glu Ile 1 5 1015 333 35 PRT Homo sapiens 333 Met Leu Ala Phe Ser His Trp Thr Tyr GluTyr Thr Arg Gly Glu Leu 1 5 10 15 Leu Val Leu Asp Leu Gln Gly Val GlyGlu Asn Leu Thr Asp Pro Ser 20 25 30 Val Ile Lys 35 334 22 PRT Homosapiens 334 Ala Lys His His Cys Asn Ser Cys Cys Arg Lys Leu Lys Leu ProAsp 1 5 10 15 Leu Lys Arg Asn Asp Tyr 20 335 20 PRT Homo sapiens 335 GluTyr Thr Arg Gly Glu Leu Leu Val Leu Asp Leu Gln Gly Val Gly 1 5 10 15Glu Asn Leu Thr 20

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95.0% identical toa sequence selected from the group consisting of: (a) a polynucleotidefragment of SEQ ID NO:1 or a polynucleotide fragment of the cDNAsequence included in ATCC Deposit No: PTA-4175, which is hybridizable toSEQ ID NO 1; (b) a polynucleotide encoding a polypeptide fragment of SEQID NO:2 or a polypeptide fragment encoded by the cDNA sequence includedin ATCC Deposit No: PTA-4175, which is hybridizable to SEQ ID NO:1; (c)a polynucleotide encoding a polypeptide domain of SEQ ID NO:2 or apolypeptide domain encoded by the cDNA sequence included in ATCC DepositNo: PTA-4175, which is hybridizable to SEQ ID NO:1; (d) a polynucleotideencoding a polypeptide epitope of SEQ ID NO:2 or a polypeptide epitopeencoded by the cDNA sequence included in ATCC Deposit No: PTA-4175,which is hybridizable to SEQ ID NO:1; (e) a polynucleotide encoding apolypeptide of SEQ ID NO:2 or the cDNA sequence included in ATCC DepositNo: PTA-4175, which is hybridizable to SEQ ID NO:1, having transientpotential protein activity; (f) an isolated polynucleotide comprisingnucleotides 4 to 6051 of SEQ ID NO:1, wherein said nucleotides encode apolypeptide corresponding to amino acids 2 to 2017 of SEQ ID NO:2 minusthe start codon; (g) an isolated polynucleotide comprising nucleotides 1to 6051 of SEQ ID NO:1, wherein said nucleotides encode a polypeptidecorresponding to amino acids 1 to 2017 of SEQ ID NO:2 including thestart codon; (h) an isolated polynucleotide comprising nucleotides 904to 1064 of SEQ ID NO:1, wherein said nucleotides encode an ion transportdomain of the TRP-PLIK2 polypeptide of SEQ ID NO:2; (j) a polynucleotidewhich represents the complimentary sequence (antisense) of SEQ ID NO:1;(k) a polynucleotide fragment of SEQ ID NO:3 which is hybridizable toSEQ ID NO:3; (l) a polynucleotide encoding a polypeptide fragment of SEQID NO:4 which is hybridizable to SEQ ID NO:3; (m) a polynucleotideencoding a polypeptide domain of SEQ ID NO:4 which is hybridizable toSEQ ID NO:3; (n) a polynucleotide encoding a polypeptide epitope of SEQID NO:4 which is hybridizable to SEQ ID NO:3; (o) a polynucleotideencoding a polypeptide of SEQ ID NO:4 which is hybridizable to SEQ IDNO:3, having transient potential protein activity; (p) an isolatedpolynucleotide comprising nucleotides 4 to 5910 of SEQ ID NO:3, whereinsaid nucleotides encode a polypeptide corresponding to amino acids 2 to1970 of SEQ ID NO:4 minus the start codon; (q) an isolatedpolynucleotide comprising nucleotides 1 to 5910 of SEQ ID NO:3, whereinsaid nucleotides encode a polypeptide corresponding to amino acids 1 to1970 of SEQ ID NO:4 including the start codon; (r) an isolatedpolynucleotide comprising nucleotides 857 to 1012 of SEQ ID NO:3,wherein said nucleotides encode an ion transport domain of theTRP-PLIK2b polypeptide of SEQ ID NO:4; (s) a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:3; (t) apolynucleotide fragment of SEQ ID NO:5 which is hybridizable to SEQ IDNO:5; (u) a polynucleotide encoding a polypeptide fragment of SEQ IDNO:6 which is hybridizable to SEQ ID NO:5; (v) a polynucleotide encodinga polypeptide domain of SEQ ID NO:6 which is hybridizable to SEQ IDNO:5; (w) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:6which is hybridizable to SEQ ID NO:5; (x) a polynucleotide encoding apolypeptide of SEQ ID NO:6 which is hybridizable to SEQ ID NO:5, havingtransient potential protein activity; (y) an isolated polynucleotidecomprising nucleotides 4 to 5817 of SEQ ID NO:5, wherein saidnucleotides encode a polypeptide corresponding to amino acids 2 to 1939of SEQ ID NO:6 minus the start codon; (z) an isolated polynucleotidecomprising nucleotides 1 to 5817 of SEQ ID NO:5, wherein saidnucleotides encode a polypeptide corresponding to amino acids 1 to 1939of SEQ ID NO:6 including the start codon; (aa) an isolatedpolynucleotide comprising nucleotides 826 to 986 of SEQ ID NO:5, whereinsaid nucleotides encode an ion transport domain of the TRP-PLIK2cpolypeptide of SEQ ID NO:6; (bb) a polynucleotide which represents thecomplimentary sequence (antisense) of SEQ ID NO:5; (cc) a polynucleotidefragment of SEQ ID NO:7 which is hybridizable to SEQ ID NO:7; (dd) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:8 which ishybridizable to SEQ ID NO:7; (ee) a polynucleotide encoding apolypeptide domain of SEQ ID NO:8 which is hybridizable to SEQ ID NO:7;(ff) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:8which is hybridizable to SEQ ID NO:7; (gg) a polynucleotide encoding apolypeptide of SEQ ID NO:8 which is hybridizable to SEQ ID NO:7, havingtransient potential protein activity; (hh) an isolated polynucleotidecomprising nucleotides 4 to 5922 of SEQ ID NO:7, wherein saidnucleotides encode a polypeptide corresponding to amino acids 2 to 1974of SEQ ID NO:8 minus the start codon; (ii) an isolated polynucleotidecomprising nucleotides 1 to 5922 of SEQ ID NO:7, wherein saidnucleotides encode a polypeptide corresponding to amino acids 1 to 1974of SEQ ID NO:8 including the start codon; (jj) an isolatedpolynucleotide comprising nucleotides 904 to 959 of SEQ ID NO:7, whereinsaid nucleotides encode an ion transport domain of the TRP-PLIK2dpolypeptide of SEQ ID NO:8; (kk) a polynucleotide which represents thecomplimentary sequence (antisense) of SEQ ID NO:7; (ll) a polynucleotidefragment of SEQ ID NO:97 which is hybridizable to SEQ ID NO:97; (mm) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:98 which ishybridizable to SEQ ID NO:97; (nn) a polynucleotide encoding apolypeptide domain of SEQ ID NO:98 which is hybridizable to SEQ IDNO:97; (oo) a polynucleotide encoding a polypeptide epitope of SEQ IDNO:98 which is hybridizable to SEQ ID NO:97; (pp) a polynucleotideencoding a polypeptide of SEQ ID NO:98 which is hybridizable to SEQ IDNO:97, having transient potential protein activity; (qq) an isolatedpolynucleotide comprising nucleotides 29 to 1513 of SEQ ID NO:97,wherein said nucleotides encode a polypeptide corresponding to aminoacids 2 to 496 of SEQ ID NO:98 minus the start codon; (rr) an isolatedpolynucleotide comprising nucleotides 1 to 1513 of SEQ ID NO:97, whereinsaid nucleotides encode a polypeptide corresponding to amino acids 1 to496 of SEQ ID NO:98 including the start codon; (ss) a polynucleotidewhich represents the complimentary sequence (antisense) of SEQ ID NO:97;and (tt) a polynucleotide capable of hybridizing under stringentconditions to any one of the polynucleotides specified in (a)-(ss),wherein said polynucleotide does not hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequence ofonly A residues or of only T residues.
 2. The isolated nucleic acidmolecule of claim 1, wherein the polynucleotide fragment comprises anucleotide sequence encoding a human transient potential receptorprotein.
 3. A recombinant vector comprising the isolated nucleic acidmolecule of claim
 1. 4. A recombinant host cell comprising the vectorsequences of claim
 3. 5. An isolated polypeptide comprising an aminoacid sequence at least 95.0% identical to a sequence selected from thegroup consisting of: (a) a polypeptide fragment of SEQ ID NO:2 or theencoded sequence included in ATCC Deposit No: PTA-4175; (b) apolypeptide fragment of SEQ ID NO:2 or the encoded sequence included inATCC Deposit No: PTA-4175, having transient potential protein activity;(c) a polypeptide domain of SEQ ID NO:2 or the encoded sequence includedin ATCC Deposit No: PTA-4175; (d) a polypeptide epitope of SEQ ID NO:2or the encoded sequence included in ATCC Deposit No: PTA-4175; (e) afull length protein of SEQ ID NO:2 or the encoded sequence included inATCC Deposit No: PTA-4175; (f) a polypeptide comprising amino acids 2 to2017 of SEQ ID NO:2, wherein said amino acids 2 to 2017 comprise apolypeptide of SEQ ID NO:2 minus the start methionine; (g) a polypeptidecomprising amino acids 1 to 2017 of SEQ ID NO:2; (h) a polypeptideencoded by polynucleotides 904 to 1064 of SEQ ID NO:1, wherein saidnucleotides encode an ion transport domain of the TRP-PLIK2 polypeptideof SEQ ID NO:2; (i) a polypeptide fragment of SEQ ID NO:4; (j) apolypeptide fragment of SEQ ID NO:4, having transient potential proteinactivity; (k) a polypeptide domain of SEQ ID NO:4; (l) a polypeptideepitope of SEQ ID NO:4; (m) a full length protein of SEQ ID NO:4; (n) apolypeptide comprising amino acids 2 to 1970 of SEQ ID NO:4, whereinsaid amino acids 2 to 1970 comprise a polypeptide of SEQ ID NO:4 minusthe start methionine; (o) a polypeptide comprising amino acids 1 to 1970of SEQ ID NO:4; (p) a polypeptide encoded by polynucleotides 857 to 1012of SEQ ID NO:3, wherein said nucleotides encode an ion transport domainof the TRP-PLIK2 polypeptide of SEQ ID NO:4; (q) a polypeptide fragmentof SEQ ID NO:6; (r) a polypeptide fragment of SEQ ID NO:6, havingtransient potential protein activity; (s) a polypeptide domain of SEQ IDNO:6; (t) a polypeptide epitope of SEQ ID NO:6; (u) a full lengthprotein of SEQ ID NO:6; (v) a polypeptide comprising amino acids 2 to1939 of SEQ ID NO:6, wherein said amino acids 2 to 1939 comprise apolypeptide of SEQ ID NO:6 minus the start methionine; (w) a polypeptidecomprising amino acids 1 to 1939 of SEQ ID NO:6; (x) a polypeptideencoded by polynucleotides 826 to 986 of SEQ ID NO:5, wherein saidnucleotides encode an ion transport domain of the TRP-PLIK2 polypeptideof SEQ ID NO:6; (y) a polypeptide fragment of SEQ ID NO:8; (z) apolypeptide fragment of SEQ ID NO:8, having transient potential proteinactivity; (aa) a polypeptide domain of SEQ ID NO:8; (bb) a polypeptideepitope of SEQ ID NO:8; (cc) a full length protein of SEQ ID NO:8; (dd)a polypeptide comprising amino acids 2 to 1974 of SEQ ID NO:8, whereinsaid amino acids 2 to 1974 comprise a polypeptide of SEQ ID NO:8 minusthe start methionine; (ee) a polypeptide comprising amino acids 1 to1974 of SEQ ID NO:8; (ff) a polypeptide encoded by polynucleotides 904to 959 of SEQ ID NO:7, wherein said nucleotides encode an ion transportdomain of the TRP-PLIK2 polypeptide of SEQ ID NO:8; (gg) a polypeptidefragment of SEQ ID NO:98; (hh) a polypeptide fragment of SEQ ID NO:98,having transient potential protein activity; (ii) a polypeptide domainof SEQ ID NO:98; (jj) a polypeptide epitope of SEQ ID NO:98; (kk) a fulllength protein of SEQ ID NO:98; (ll) a polypeptide comprising aminoacids 2 to 496 of SEQ ID NO:98, wherein said amino acids 2 to 496comprise a polypeptide of SEQ ID NO:98 minus the start methionine; and(mm) a polypeptide comprising amino acids 1 to 496 of SEQ ID NO:98. 6.The isolated polypeptide of claim 5, wherein the full length proteincomprises sequential amino acid deletions from either the C-terminus orthe N-terminus.
 7. An isolated antibody that binds specifically to theisolated polypeptide of claim
 5. 8. A recombinant host cell thatexpresses the isolated polypeptide of claim
 5. 9. A method of making anisolated polypeptide comprising: (a) culturing the recombinant host cellof claim 8 under conditions such that said polypeptide is expressed; and(b) recovering said polypeptide.
 10. The polypeptide produced by claim9.
 11. A method for preventing, treating, or ameliorating a medicalcondition, comprising the step of administering to a mammalian subject atherapeutically effective amount of the polypeptide of claim
 5. 12. Amethod of diagnosing a pathological condition or a susceptibility to apathological condition in a subject comprising: (a) determining thepresence or absence of a mutation in the polynucleotide of claim 1; and(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.
 13. A method of diagnosing a pathological condition or asusceptibility to a pathological condition in a subject comprising: (a)determining the presence or amount of expression of the polypeptide ofclaim 5 in a biological sample; and (b) diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thepresence or amount of expression of the polypeptide.
 14. An isolatednucleic acid molecule consisting of a polynucleotide having a nucleotidesequence selected from the group consisting of: (a) a polynucleotideencoding a polypeptide of SEQ ID NO:2; (b) an isolated polynucleotideconsisting of nucleotides 4 to 6051 of SEQ ID NO:1, wherein saidnucleotides encode a polypeptide corresponding to amino acids 2 to 2017of SEQ ID NO:2 minus the start codon; (c) an isolated polynucleotideconsisting of nucleotides 1 to 6051 of SEQ ID NO:1, wherein saidnucleotides encode a polypeptide corresponding to amino acids 1 to 2017of SEQ ID NO:2 including the start codon; (d) a polynucleotide encodingthe TRP-PLIK2 polypeptide encoded by the cDNA clone contained in ATCCDeposit No. PTA-4175; (e) an isolated polynucleotide consisting ofnucleotides 904 to 1064 of SEQ ID NO:1, wherein said nucleotides encodean ion transport domain of the TRP-PLIK2 polypeptide of SEQ ID NO:2; (f)a polynucleotide which represents the complimentary sequence (antisense)of SEQ ID NO:1; (g) a polynucleotide encoding a polypeptide of SEQ IDNO:4; (h) an isolated polynucleotide consisting of nucleotides 4 to 5910of SEQ ID NO:3, wherein said nucleotides encode a polypeptidecorresponding to amino acids 2 to 1970 of SEQ ID NO:4 minus the startcodon; (i) an isolated polynucleotide consisting of nucleotides 1 to5910 of SEQ ID NO:3, wherein said nucleotides encode a polypeptidecorresponding to amino acids 1 to 1970 of SEQ ID NO:4 including thestart codon; (j) an isolated polynucleotide consisting of nucleotides857 to 1012 of SEQ ID NO:3, wherein said nucleotides encode an iontransport domain of the TRP-PLIK2bpolypeptide of SEQ ID NO:4; (k) apolynucleotide which represents the complimentary sequence (antisense)of SEQ ID NO:3; (l) a polynucleotide encoding a polypeptide of SEQ IDNO:6; (m) an isolated polynucleotide consisting of nucleotides 4 to 5817of SEQ ID NO:5, wherein said nucleotides encode a polypeptidecorresponding to amino acids 2 to 1939 of SEQ ID NO:6 minus the startcodon; (n) an isolated polynucleotide consisting of nucleotides 1 to5817 of SEQ ID NO:5, wherein said nucleotides encode a polypeptidecorresponding to amino acids 1 to 1939 of SEQ ID NO:6 including thestart codon; (o) an isolated polynucleotide consisting of nucleotides826 to 1939 of SEQ ID NO:5, wherein said nucleotides encode an iontransport domain of the TRP-PLIK2c polypeptide of SEQ ID NO:6; (p) apolynucleotide which represents the complimentary sequence (antisense)of SEQ ID NO:5; (q) a polynucleotide encoding a polypeptide of SEQ IDNO:8; (r) an isolated polynucleotide consisting of nucleotides 4 to 5922of SEQ ID NO:7, wherein said nucleotides encode a polypeptidecorresponding to amino acids 2 to 1974 of SEQ ID NO:8 minus the startcodon; (s) an isolated polynucleotide consisting of nucleotides 1 to5922 of SEQ ID NO:7, wherein said nucleotides encode a polypeptidecorresponding to amino acids 1 to 1974 of SEQ ID NO:8 including thestart codon; (t) an isolated polynucleotide consisting of nucleotides904 to 959 of SEQ ID NO:7, wherein said nucleotides encode an iontransport domain of the TRP-PLIK2d polypeptide of SEQ ID NO:8; (u) apolynucleotide which represents the complimentary sequence (antisense)of SEQ ID NO:7; (v) a polynucleotide encoding a polypeptide of SEQ IDNO:98; (w) an isolated polynucleotide consisting of nucleotides 29 to1513 of SEQ ID NO:97, wherein said nucleotides encode a polypeptidecorresponding to amino acids 2 to 496 of SEQ ID NO:98 minus the startcodon; (x) an isolated polynucleotide consisting of nucleotides 1 to1513 of SEQ ID NO:97, wherein said nucleotides encode a polypeptidecorresponding to amino acids 1 to 496 of SEQ ID NO:98 including thestart codon; and (y) a polynucleotide which represents the complimentarysequence (antisense) of SEQ ID NO:97.
 15. The isolated nucleic acidmolecule of claim 14, wherein the polynucleotide comprises a nucleotidesequence encoding a human transient potential receptor protein.
 16. Arecombinant vector comprising the isolated nucleic acid molecule ofclaim
 15. 17. A recombinant host cell comprising the recombinant vectorof claim
 16. 18. An isolated polypeptide consisting of an amino acidsequence selected from the group consisting of: (a) a polypeptidefragment of SEQ ID NO:2 having transient potential protein activity; (b)a polypeptide domain of SEQ ID NO:2 having transient potential proteinactivity; (c) a full length protein of SEQ ID NO:2; (d) a polypeptidecorresponding to amino acids 2 to 2017 of SEQ ID NO:2, wherein saidamino acids 2 to 2017 consisting of a polypeptide of SEQ ID NO:2 minusthe start methionine; (e) a polypeptide corresponding to amino acids 1to 2017 of SEQ ID NO:2; (f) a polypeptide encoded by the cDNA containedin ATCC Deposit No. PTA-4175; (g) a polypeptide encoded bypolynucleotides 904 to 1064 of SEQ ID NO:1, wherein said nucleotidesencode an ion transport domain of the TRP-PLIK2 polypeptide of SEQ IDNO:2; (h) a polypeptide fragment of SEQ ID NO:4 having transientpotential protein activity; (i) a polypeptide domain of SEQ ID NO:4having transient potential protein activity; (j) a full length proteinof SEQ ID NO:4; (k) a polypeptide corresponding to amino acids 2 to 1970of SEQ ID NO:4, wherein said amino acids 2 to 1970 consisting of apolypeptide of SEQ ID NO:4 minus the start methionine; (l) a polypeptidecorresponding to amino acids 1 to 1970 of SEQ ID NO:4; (m) a polypeptideencoded by polynucleotides 857 to 1012 of SEQ ID NO:3, wherein saidnucleotides encode an ion transport domain of the TRP-PLIK2b polypeptideof SEQ ID NO:4; (n) a polypeptide fragment of SEQ ID NO:6 havingtransient potential protein activity; (o) a polypeptide domain of SEQ IDNO:6 having transient potential protein activity; (p) a full lengthprotein of SEQ ID NO:6; (q) a polypeptide corresponding to amino acids 2to 1939 of SEQ ID NO:6, wherein said amino acids 2 to 1939 consisting ofa polypeptide of SEQ ID NO:6 minus the start methionine; (r) apolypeptide corresponding to amino acids 1 to 1939 of SEQ ID NO:6; (s) apolypeptide encoded by polynucleotides 826 to 986 of SEQ ID NO:5,wherein said nucleotides encode an ion transport domain of theTRP-PLIK2c polypeptide of SEQ ID NO:6; (t) a polypeptide fragment of SEQID NO:8 having transient potential protein activity; (u) a polypeptidedomain of SEQ ID NO:8 having transient potential protein activity; (v) afull length protein of SEQ ID NO:8; (w) a polypeptide corresponding toamino acids 2 to 1974 of SEQ ID NO:8, wherein said amino acids 2 to 1974consisting of a polypeptide of SEQ ID NO:8 minus the start methionine;(x) a polypeptide corresponding to amino acids 1 to 1974 of SEQ ID NO:8;(y) a polypeptide encoded by polynucleotides 904 to 959 of SEQ ID NO:7,wherein said nucleotides encode an ion transport domain of theTRP-PLIK2d polypeptide of SEQ ID NO:8; (z) a polypeptide fragment of SEQID NO:98 having transient potential protein activity; (aa) a polypeptidedomain of SEQ ID NO:98 having transient potential protein activity; (bb)a full length protein of SEQ ID NO:98; (cc) a polypeptide correspondingto amino acids 2 to 496 of SEQ ID NO:98, wherein said amino acids 2 to496 consisting of a polypeptide of SEQ ID NO:98 minus the startmethionine; and (dd) a polypeptide corresponding to amino acids 1 to 496of SEQ ID NO:98.
 19. The method for preventing, treating, orameliorating a medical condition of claim 11, wherein the medicalcondition is selected from the group consisting of an immune disorder, ahematopoietic disorder, an inflammatory disorder, a renal disorder, areproductive disorder, a hepatic disorder, a disorder related to hypertransient receptor potential activity, prostate cancer, testicularcancer, diseases related to chromosome 9q21.2-22.1 aberrations,amyotrophic lateral sclerosis with frontotemporal dementia, early-onsetpulverulent cataract, infantile nephronophthisis, hypomagnesemia withsecondary hypocalcemia familial hemophagocytic lymphohistiocytosis,neuronal degeneration, neurogenic inflammation, allergy,immunodeficiency/excessive immune activation, visual defects, hearingdisorder, pain, cancer, hypertension, cardiovascular diseases, diseasesassociated with disturbances in Ca²⁺ homeostasis including osteoporosis,hypercalciuric stone disease, chronic renal failure, proliferativedisorders, cancers, ischemia-reperfusion injury, heart failure, immunocompromised conditions, HIV infection, disorders associated withaberrant NFkB regulation, disorders associated with aberrant apoptosisregulation, disorders in which decreasing NFKB activity would betherapeutically desirable, disorders in which increasing NFKB activitywould be therapeutically desirable, disorders in which decreasing IkBactivity would be therapeutically desirable, and disorders in whichincreasing IkB activity would be therapeutically desirable.